Polypeptides of fusobacterium and methods of use

ABSTRACT

The present invention provides isolated polypeptides isolatable from a  Fusobacterium  spp. Also provided by the present invention are compositions that include one or more of the polypeptides, and methods for making and methods for using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/252,951, filed Nov. 9, 2015, which is incorporated by referenceherein.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submittedvia EFS-Web to the United States Patent and Trademark Office as an ASCIItext file entitled “29300540201 SequenceListing_ST25.txt” having a sizeof 339 kilobytes and created on Nov. 9, 2016. The information containedin the Sequence Listing is incorporated by reference herein.

BACKGROUND

Fusobacterium spp. are gram-negative, obligately anaerobic andpleomorphically rod shaped bacterium responsible for a variety ofnecrotic infections in animals and in humans (Langworth, Bacteriol.Rev., 41, 373-390 (1977)). Pathogenic species in the Genus Fusobacteriuminclude F. necrophorum, F. nucleatum, F. canifelinum, F. gonidiaformans,F. mortiferum, F. naviforme, F. necrogenes, F. russii, F. ulcerans, andF. varium. Fusobacterium necrophorum is the most pathogenic and isclassified into two subspecies: F. necrophorum subsp. necrophorum and F.necrophorum subsp. funduliforme and are responsible for a number ofclinical manifestations in various species of animals, such as cattle,horses, goats, sheep, fowl, and swine, including hepatic abscesses, footrot, laminitis, purulent and interdigital dermatitis, contagiousecthyma, necrotic rhinitis, and necrotic laryngitis. Taxa formally inthe genus Fusobacterium include Fillfactor alocis, commonly found in theperiodontal pockets of patients having periodontitis (Kumar et al.,2003, J Dent Res., 82(5):338-44); Faecalibacterium prausnitzii, andEubacterium sulci, also associated with odontogenic infections (Munsonet al., 2002, J. Dent Res., 81:761, Paster et al., 2006, Periodontology2000, 42:80). Although the primary etiologic agent of liver abscesseshas been shown to be Fusobacterium necrophorum abscessation has beenassociated with other bacteriological agents such as Arcanobacteriumpyogenes. Bacteroides spp., Salmonella spp., Clostridium spp.,Pasteurella spp., E. coli spp., and Peptostreptococcus spp.

In humans, F. necrophorum and F. nucleatum are considered to be the mostpathogenic and is the causative agent of skin ulcers, peritonsillarabscesses, septic arthritis, Lemierre's syndrome, periodontal diseases,endocarditis and metastatic abscesses in the lungs, liver, joints, andpleural spaces. On a population-based perspective Fusobacteriumbacteremia is relatively uncommon in humans with an overall annualincidence of 0.55 per 100,000 population. The incidence of F. nucleatumwas found to be 0.34/100,000 and F. necrophorum was 0.14/100,000 with amedian age of 53.5 years while F. necrophorum cases had a median age of21 years. Overall mortality due to bacteremia was 11 percent. F.necrophorum affects mostly young health adults. In contrast, F.nucleatum affects older individuals with seemingly compromised healthyconditions (Afra, Infectious Diseases, 13: 264 (2013)). A number ofother species of fusobacteria have been implicated as the etiologicalagent in a variety of diseases, for example, F. ulcercans (skin ulcers),F. russi (animal bite infections), and F. varium (eye infections) (Smithet al., Epidemiol Infect., 110, 499-506 (1993)).

In beef-breed and Holstein steers, the incidence of liver abscessesaverage from 12 to 32% in most feedlots, and has been shown to beinfluenced by a number of dietary and management factors. Liverabscesses are categorized as mild, moderate or severe. Severe liverlesions are most often associated with high economic losses toproducers, packers, and ultimately consumers. Besides livercondemnation, economic impacts include reduced feed intake, reducedweight gain, decreased feed efficiency, and decreased carcass yield.

F. necrophorum possesses a number of virulence factors that participatein the penetration and colonization of the ruminal epithelium andsubsequent entry and establishment of infection in the liver, includinga potent secreted leukotoxin which has been shown to be specificallytoxic to ruminant polymorphonuclear leukocytes (Tan et al., Vet. Res.Commun. 20, 113-140 (1996)). The role of leukotoxin as a virulencefactor has been documented. For instance, experiments have indicated acorrelation between toxin production and the ability of F. necrophorumto induce abscesses in laboratory animals (Coyle et al., Am. J. Vet.Res., 40, 274-276. (1979), and Tan et al., Am. J. Vet. Res., 55, 515.(1994)). Experiments have also shown that non-leukotoxin producingstrains are unable to induce foot abscesses in cattle followingchallenge. It has also been shown that neutralizing antibody produced byan inactivated toxoid derived from leukotoxin reduced infection andliver abscesses in vaccinated cattle.

Control of liver abscesses in feedlot cattle generally has depended onthe use of antimicrobial compounds. Five antibiotics (i.e., bacitracinmethylene disalicylate, chlortetracycline, oxytetracycline, tylosin, andvirginiamycin) are approved for prevention of liver abscesses in feedlotcattle. Tylosin is the most effective and the most commonly used feedadditive.

A number of commercial killed whole cell bacterins have been used tocontrol necrotic infection in farm animals incorporating multiplestrains including the most prevalent serotypes such as biotype A (F.necrophorum subsp. necrophorum). Another approach to vaccine developmenthas been the incorporation of leukotoxin as a toxoid to prevent thepathological effect of the secreted toxin (Saginala et al., J. Anim.Sci., 75, 11601166 (1997)).

Divalent metal ions such as iron, cobalt, copper, magnesium, manganese,molybdenum, nickel, selenium, and zinc and are trace elements oftenrequired for the survival of bacteria infecting both animal and humanhosts. These trace metal elements are used by bacteria as cofactors forenzymes that catalyze biochemical reactions for various metabolicpathways required by the organism. The impact of iron on thepathogenesis of bacteria has been studied extensively. Iron is essentialfor nearly all life and is required for enzymatic and metabolic pathwaysof cells at all phylogenic levels. It has been well-documented thatduring bacterial sepsis there is an alteration in the concentration of anumber of metal ions in serum such as, iron, copper, and zinc. Forinstance, serum levels of zinc decrease from 10 percent to 60 percentwith the onset of infection. Following the onset of infection, zinc isthen redistributed from plasma to liver where it is bound tometallothionein. Decreases in serum iron of up to 50 percent have beendescribed during infectious illness, whereas serum copper has been shownto increase in response to inflammatory stimuli. The alteration of thesetrace metal ions in serum may directly affect the severity orprogression of any bacterial infection.

The ability of Fusobacterium to evade the natural defense mechanisms ofthe vertebrate host depends in part on its ability to obtain host iron,which in turn, directly influences the host-pathogen interaction.Because of iron's essential nature, vertebrate hosts have developedelaborate mechanisms to bind iron in body fluids (e.g., transferrin inblood and lymph fluids and lactoferrin in external secretions). Thesehigh affinity iron binding proteins create an iron restrictedenvironment within the host, reducing the level of iron to approximately10⁻¹⁸ molar, a concentration too low to support the growth of nearly allbacteria. These iron sequestering mechanisms of the host act as anatural defense mechanism to combat bacterial invasion. To circumventthese iron-restrictive conditions many bacterial species have evolvedmechanisms for obtaining iron. The most common mechanisms include thediffusion of soluble iron through porins and specialized transportsystems that mediate the uptake of iron by siderophores. This lattersystem is one of the most common and well-studied mechanisms for ironacquisition and involves the specific chelation of ferric iron bysiderophores and the synthesis of their cognate transport systems, whichpermits the bacteria to continue to replicate and overcome thenon-specific defense mechanisms of the host. Continued replication, andthus each step in the infectious process, is ultimately dependent on theability of the organism to obtain iron from its host.

With so many basic functions relying on the availability of iron,bacteria have evolved a complex regulatory network for acquiring ironunder varying physiological conditions. Under anaerobic conditions, ironis present in the soluble ferrous form (Fe II) and can freely diffusethrough outer membrane porins into the periplasm. For instance, in E.coli the FeoAB transport system present in the cytoplasmic membrane willtransport the ferrous iron molecules into the cell cytoplasm. Underaerobic conditions and neutral pH, iron is primarily present in theinsoluble ferric form (Fe III) and cannot pass through the outermembrane porins by passive diffusion. Instead, molecules calledsiderophores are secreted by bacteria, which have a high affinity forferric iron. The ferric-siderophore complexes are recognized byreceptors in the outer membrane collectively referred to as theTonB-dependent receptors. These receptors, once bound to loadedsiderophores, are believed to interact with TonB and its associatedproteins localized in the periplasm and cytoplasmic membrane. Theseprotein-protein interactions, though poorly understood, serve to providethe energy necessary to transport the ferri-siderophore complexes acrossthe outer membrane and through the periplasmic space. ABC transportsystems present in the cytoplasmic membrane serve to transport theiron-siderophore complexes across the cytoplasmic membrane. Reductaseenzymes reduce the ferric iron to its ferrous form, which dissociates itfrom the siderophore and releases iron into the cell.

Several species of pathogenic bacteria use additional mechanisms toobtain iron from mammalian hosts, including the direct binding oftransferrin, heme, and other heme-containing compounds. The receptorproteins that bind these iron-containing molecules most likely rely onthe TonB complex for the energy required to transport heme across theouter membrane, similar to the iron-siderophore complexes. SpecializedABC transporters are then used to transport the heme across thecytoplasmic membrane. In addition, some bacteria secrete hemophores,small molecules that can bind heme and present it to receptors on thebacterial cell surface. Several pathogenic species also producehemolysins, which are toxins that lyse red blood cells, releasing hemeand hemoglobin for uptake by the bacteria.

The outer membrane proteins of gram-negative bacteria control theselective permeability of many essential nutrients critical to thesurvival of bacteria, including all pathogenic bacteria that causedisease in animals and man. This selective permeability of nutrients iscontrolled by a class of membrane proteins called porins. It now appearsthat the majority of the outer membrane proteins on the surface ofgram-negative bacteria are porins, identified as the general porins(e.g., OmpF), monomeric porins (e.g., OmpA), the specific porins (e.g.,the maltose-specific porin LamB) and the TonB-dependent, gated porins(e.g., the siderophore receptor FepA). The porin class of proteinsgenerally share structural features, including the presence ofbeta-barrels that span the outer membrane.

Little is known regarding the iron-acquisition by Fusobacterium spp, andgenomic comparisons are difficult since the genome of only five strainsof Fusobacterium nucleatum have been completely sequenced and madepublicly available: F. nucleatum subspecies nucleatum, strain ATCC 25586(Kapatral et al., J. Bacteriol., 184, 2005-2018 (2002)); Fusobacteriumnucleatum subsp. vincentii 3_1_36A2; Fusobacterium nucleatum subsp.vincentii 3_1_36A2; Fusobacterium nucleatum subsp. animalis 7_1; andFusobacterium nucleatum subsp. animalis 4_8 (Tatusova T, et al. NucleicAcids Res 42, D553-D559 (2014). No complete sequence of Fusobacteriumnecrophorum strains have been published, although there are a number ofpartial sequences in the NCBI database The genomic sequence of ATCC25586 was used in a comparison with a partially sequenced genome of F.nucleatum subspp. vincentii (Kapatral et al., Genome Res., 13, 1180-1189(2003)) to investigate differences among these two subspecies. Theresults suggested that there were differences between the two genomeswith respect to the iron uptake systems. Although iron transport systemswere discovered in both genomes, the genome of strain ATCC 25586contains three additional iron-specific ABC transport systems. Inaddition, hemin receptor proteins appear to be encoded by both genomes,but while the subspp. vincentii isolate encodes three receptors, thegenome of strain ATCC 25586 apparently encodes five such proteins.Furthermore, the feoAB genes, encoding a putative ferrous iron transportsystem, are only found in the genome of the subspp. vincentii isolate.Since both organisms are obligate anaerobes and ferrous iron is thepredominant form of the metal under anaerobic conditions, strain ATCC25586 may have a second mechanism for uptake of ferrous iron. Given thedifferences among these two subspecies of F. nucleatum, it is likelythat there will be many differences among the iron uptake systemsbetween other Fusobacterium species. Therefore, the F. nucleatum genomicdata may not be useful for predicting the presence or absence of ironacquisition systems in other species of Fusobacterium.

Fusobacterium necrophorum is ubiquitous in the environment of cattle andis considered a normal inhabitant of the intestine and rumen, and ispresent in feces. The organism is the causative agent of both liverabscesses and footrot. The disease is rarely fatal but can result insubstantial economic losses to both the producer and packer due to costin treatment and performance losses. It is thought that liverabscessation follows a condition of ruminal acidosis which impairs theintegrity of the rumen wall allowing Fusobacterium to transverse to theblood stream to the liver and cause abscessation.

Beyond the role of iron as an essential nutrient for microbial survival,there are now many other well-defined transitional metals that playcritical roles in bacterial survival, homeostasis and pathogenesis suchas iron, manganese, copper, zinc, magnesium, cobalt, and nickel (Waldronand Robinson, 2009, Nature Reviews Microbiology, 7:25-35; Porcheron,2013, Frontiers in Cellular and Infection Microbiology 3:172-194). Iron,zinc and copper are the three most abundant divalent metal ions inmammals in descending order of concentration. The ability of a bacteriumto use these transitional metals by finely regulated uptake oracquisition systems significantly contributes to the virulence ofpathogenic bacteria. It is well known that bacteria within the samegenus/species do not have the same uptake systems for the acquisition oftransitional metals owing to the difference in pathogenicity from onestrain of bacteria to another. These differences in the ability ofbacteria to use different transitional metals based on expressed uptakesystems may specifically direct what organ or tissue an organism caninvade.

Copper is the third most prevalent transitional metal behind iron andzinc and plays a major role in many enzymatic pathways. Copper ispresent in every tissue of the body but is stored in its highestconcentration in the liver. What role copper plays in the virulence ofFusobacterium is unknown. With the concentration of copper being thehighest in the liver it would make sense that Fusobacterium has adaptedsome mechanism to utilize this divalent metal ion once in the liver.

In the following examples we show the expression of unique proteins thatare expressed in Fusobacterium when grown under iron, zinc and copperchelation.

SUMMARY OF THE INVENTION

Provided herein are compositions. In one embodiment, a compositionincludes at least one isolated polypeptide having a molecular weight of92 kDa to 79 kDa, 73 kDa to 63 kDa, 62 kDa to 58 kDa, or 57 kDa to 47kDa, wherein the at least one polypeptide is isolatable from aFusobacterium necrophorum when incubated in media including an ironchelator and not isolatable when grown in the media without the ironchelator; at least one isolated polypeptide having a molecular weight of108 kDa to 98 kDa or 79 kDa to 69 kDa, wherein the at least onepolypeptide is isolatable from a Fusobacterium necrophorum whenincubated in media including an iron chelator, is expressed by theFusobacterium necrophorum when incubated in media without the ironchelator and expressed at an enhanced level during growth in mediaincluding an iron chelator; and at least one protein selected from thegroup consisting of a polypeptide having at least 85% similarity to SEQID NO:4 or a fragment thereof, a polypeptide having at least 85%similarity to SEQ ID NO:2 or a fragment thereof, a polypeptide having atleast 85% similarity to SEQ ID NO:6 or a fragment thereof, a polypeptidehaving at least 85% similarity to SEQ ID NO:34 or a fragment thereof,and a polypeptide having at least 85% similarity to SEQ ID NO:53 or afragment thereof.

In one embodiment, a composition includes isolated polypeptides havingmolecular weights of 92 kDa to 79 kDa, 73 kDa to 63 kDa, 62 kDa to 58kDa, and 57 kDa to 47 kDa, wherein the polypeptides are isolatable froma Fusobacterium necrophorum when incubated in media including an ironchelator and not isolatable when grown in the media without the ironchelator; isolated polypeptides having molecular weights of 155 kDa to145 kDa or 89 kDa to 79 kDa, wherein the at least one polypeptide isisolatable from a Fusobacterium necrophorum when incubated in mediaincluding an iron chelator and an iron-containing porphyrin and notisolatable when grown in the media without the iron chelator andiron-containing porphyrin, and not isolatable when grown in the mediawith the iron chelator and in the absence of the iron-containingporphyrin; and isolated polypeptides having molecular weights of 108 kDato 98 kDa and 79 kDa to 69 kDa, wherein the polypeptides are isolatablefrom a Fusobacterium necrophorum when incubated in media including aniron chelator, are expressed by the Fusobacterium necrophorum whenincubated in media without the iron chelator and expressed at anenhanced level during growth in media including an iron chelator.

In one embodiment, a composition includes at least one isolatedpolypeptide having a molecular weight of 131 kDa to 121 kDa, 79 kDa to69 kDa, or 33 kDa to 23 kDa, wherein the at least one polypeptide isisolatable from a Fusobacterium necrophorum when incubated in mediaincluding a copper chelator and not isolatable when grown in the mediawithout the copper chelator; and at least one isolated polypeptidehaving a molecular weight of 93 kDa to 83 kDa, 65 kDa to 55 kDa, or 52kDa to 42 kDa, wherein the at least one polypeptide is isolatable fromthe Fusobacterium necrophorum when incubated in media including a copperchelator, is expressed by the Fusobacterium necrophorum when incubatedin media without the copper chelator and expressed at an enhanced levelduring growth in media including an copper chelator.

In one embodiment, a composition includes at least one isolatedpolypeptide having a molecular weight of 131 kDa to 121 kDa, 108 kDa to98 kDa, 92 kDa to 64 kDa, 53 kDa to 43 kDa, or 33 kDa to 19 kDa, whereinthe polypeptide is isolatable from a Fusobacterium necrophorum whenincubated in media including a zinc chelator and not isolatable whengrown in the media without the zinc chelator; and at least one isolatedpolypeptide having a molecular weight of 79 kDa to 69 kDa or 65 kDa to55 kDa, wherein the at least one polypeptide is isolatable from theFusobacterium necrophorum when incubated in media including a zincchelator, is expressed by the Fusobacterium necrophorum when incubatedin media without the zinc chelator and expressed at an enhanced levelduring growth in media including the zinc chelator.

In one embodiment, a composition includes isolated polypeptides havingmolecular weights of 131 kDa to 121 kDa, 79 kDa to 69 kDa, and 33 kDa to23 kDa, wherein the polypeptides are isolatable from a Fusobacteriumnecrophorum when incubated in media including a copper chelator and notisolatable when grown in the media without the copper chelator; andisolated polypeptides having molecular weights of 93 kDa to 83 kDa, 65kDa to 55 kDa, and 52 kDa to 42 kDa, wherein the polypeptides areisolatable from the Fusobacterium necrophorum when incubated in mediaincluding a copper chelator, are expressed by the Fusobacteriumnecrophorum when incubated in media without the copper chelator andexpressed at an enhanced level during growth in media including ancopper chelator.

In one embodiment, a composition includes isolated polypeptides havingmolecular weights of 131 kDa to 121 kDa, 108 kDa to 98 kDa, 92 kDa to 64kDa, 53 kDa to 43 kDa, and 33 kDa to 19 kDa, wherein the polypeptidesare isolatable from a Fusobacterium necrophorum when incubated in mediaincluding a zinc chelator and not isolatable when grown in the mediawithout the zinc chelator; and isolated polypeptides having molecularweights of 79 kDa to 69 kDa and 65 kDa to 55 kDa, wherein thepolypeptides are isolatable from the Fusobacterium necrophorum whenincubated in media including a zinc chelator, are expressed by theFusobacterium necrophorum when incubated in media without the zincchelator and expressed at an enhanced level during growth in mediaincluding the zinc chelator.

In one embodiment, a composition can further include a polypeptidehaving at least 85% similarity to SEQ ID NO:4 or a fragment thereof, apolypeptide having at least 85% similarity to SEQ ID NO:2 or a fragmentthereof, a polypeptide having at least 85% similarity to SEQ ID NO:6 ora fragment thereof, a polypeptide having at least 85% similarity to SEQID NO:34 or a fragment thereof, a polypeptide having at least 85%similarity to SEQ ID NO:53 or a fragment thereof, or a combinationthereof.

In one embodiment, a composition includes an isolated polypeptide havingat least 85% similarity to SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, or 54, or a fragment thereof.

In one embodiment, a composition can further include isolatedpolypeptides having molecular weights of 340 kDa to 330 kDa, 247 kDa to237 kDa, 247 kDa to 237 kDa, 235 kDa to 215 kDa, 120 kDa to 110 kDa, 51kDa to 25 kDa, and 21 kDa to 11 kDa.

A composition can further include a pharmaceutically acceptable carrier,such as an adjuvant. In one embodiment, a composition protects an animalagainst challenge with Fusobacterium necrophorum.

Also provided herein are methods. In one embodiment, a method includesadministering to a subject an amount of a composition described hereineffective to induce the subject to produce antibody that specificallybinds to at least one polypeptide of the composition.

In one embodiment, a method is for treating an infection in a subject,and the method includes administering an effective amount of acomposition described herein to a subject having or at risk of having aninfection caused by a Fusobacterium spp.

In one embodiment, a method is for treating a symptom in a subject, andthe method includes administering an effective amount of a compositiondescribed herein to a subject having or at risk of having an infectioncaused by a Fusobacterium spp.

In one embodiment, a method is for decreasing colonization in a subject,and the method includes administering an effective amount of acomposition described herein to a subject colonized by a Fusobacteriumspp.

In one embodiment, a method is for treating an infection in a subject,and the method includes administering an effective amount of acomposition to a subject having or at risk of having an infection causedby a Fusobacterium spp., wherein the composition includes antibody thatspecifically binds to a polypeptide described herein. In one embodiment,a method is for treating a symptom in a subject, and the method includesadministering an effective amount of a composition to a subject havingor at risk of having an infection caused by a Fusobacterium spp.,wherein the composition includes antibody that specifically binds to apolypeptide described herein. In one embodiment, an infection causes acondition selected from metritis, hepatic abscesses, and foot rot.

In one embodiment, a method is for decreasing colonization in a subject,and the method includes administering an effective amount of acomposition to a subject colonized by a Fusobacterium spp., wherein thecomposition includes antibody that specifically binds to a polypeptidedescribed herein.

Also provided are kits. In one embodiment, a kit is for detectingantibody that specifically binds a polypeptide, including in separatecontainers an isolated polypeptide described herein, and a reagent thatdetects an antibody that specifically binds the polypeptide. In oneembodiment, a kit is for detecting a polypeptide, including in separatecontainers an antibody that specifically binds an isolated polypeptidedescribed herein, and a second reagent that specifically binds thepolypeptide.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The words “preferred” and “preferably” refer to embodiments of theinvention that may afford certain benefits, under certain circumstances.However, other embodiments may also be preferred, under the same orother circumstances. Furthermore, the recitation of one or morepreferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the invention.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

It is understood that wherever embodiments are described herein with thelanguage “include,” “includes,” or “including,” and the like, otherwiseanalogous embodiments described in terms of “consisting of” and/or“consisting essentially of” are also provided.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

Also herein, the recitations of numerical ranges by endpoints includeall numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.80, 4, 5, etc.).

For any method disclosed herein that includes discrete steps, the stepsmay be conducted in any feasible order. And, as appropriate, anycombination of two or more steps may be conducted simultaneously.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which examples can beused in various combinations. In each instance, the recited list servesonly as a representative group and should not be interpreted as anexclusive list.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows SDS-PAGE of Gel image of extracted proteins derived fromFusobacterium necrophorum grown under metal-depleted growth conditionsusing different chelators. Lane 1-Molecular Weight Marker; Lane 2-15μg/ml 2,2-dipyridyl in mTSB; Lane 3-100 μM Naringenin in mTSB; Lane4-200 μM Catechin in mTSB; Lane 5-50 μM Quercetin in mTSB; Lane 6-100 μMQuercetin in mTSB; Lane 7-50 μM TPEN in mTSB; Lane 8-50 μM ammoniumtetrathiomolybdate in mTSB and Lane 9-15 μg/ml 2,2-dipyridyl in pBHI.Lane 6 shows the expression of a novel copper protein expressed whenFusobacterium necrophorum when grown under copper chelation and lane 7shows a novel zinc protein when Fusobacterium necrophorum was grownunder zinc chelation.

FIG. 2A-2B. FIG. 2A shows SDS-PAGE gel with the banding profile ofFusobacterium necrophorum grown in mTSB containing Naringenin andCatechin. Lane 1, Molecular Weight Marker; Lane 2, 100 μM Naringenin inmTSB. Lane 3, 200 μM Catechin in mTSB. Brackets surround unique 60 kDaprotein in lane 3 resulting from growth in the presence of the metalchelator Catechin. FIG. 2B shows the corresponding Western Blot probedwith the convalescent bovine sera of Example 7. Lane 1, 100 μMNaringenin; Lane 2, 200 μM Catechin. Note the intense sero-reactive 60kDa protein of lane 2 of FIG. 2B in contrast to lane 1 of FIG. B.

FIG. 3 shows Western Blot showing the sero-reactivity of theFusobacterium necrophorum 48 kDa protein grown under copper depletegrowth conditions. Lane 1, Molecular Weight Marker (MWM); Lane2-Sero-reactivity of the 48 kDa copper protein. Arrow shows theup-regulation of a novel protein at 48 kDa that reacted with sera ofExample 7.

FIG. 4A-4B. FIG. 4A shows an SDS-PAGE gel with the banding profile ofFusobacterium necrophorum grown in mTSB containing Quercetin and TPEN.Lane 1, Molecular Weight Marker; Lane 2, 100 μM Quercetin in mTSB, Lane3, 50 μM TPEN in mTSB, Lane 4, 15 μg/ml 2,2-dipyridyl in mTSB. Bracketssurround unique 81 kDa protein up-regulated during grown in the presenceof the zinc chelator TPEN (Lane 3) compared to Lanes 2 and 4 grown withQuercetin and 2,2-dipyridyl, respectively. FIG. 4B shows thecorresponding Western Blot probed with the convalescent bovine sera ofExample 7. Lane 1,100 μM Quercetin in mTSB; Lane 2, 50 μM TPEN in mTSB,Lane 3, 15 μg/ml 2,2-dipyridyl in mTSB. Note the intense sero-reactive81 kDa protein of lane 2 grown in the presence TPEN in contrast to Lanes1 and 3 grown in Quercetin and 2,2-dipyridyl.

FIG. 5 shows the incidence of liver lesions between groups seven dayspost challenge with Fusobacterium necrophorum. All treatment groups werevaccinated two times except for Group B which received only onevaccination. There was a decrease in liver lesions between all treatmentgroups compared to controls. The only treatment group that did not showa significant difference compared to the non-vaccinated control wasGroup B.

FIG. 6 shows the difference in the size of lesions between vaccinatesand placebo controls. The lesion score was enumerated where a lesion≤0.5 cm=1(shaded boxes) and a lesion ≥0.5=2(unshaded boxes). Each barrepresents the number of challenged mice showing the total number oflesion per group that had a lesion size of ≤0.5 cm or ≥0.5.

FIG. 7A-7B. FIG. 7A shows a Western Blot of the serological response tothe rZinc protein. Lane 1, Molecular Weight Marker; Lane 2, Fuso-SRPExtract probed with sera derived from the 250 μg rZinc vaccine of GroupC; Lane 3, rZinc protein probed with sera derived from the 100 μg rZincvaccine of Group B; Lane 4, rZinc protein probed with sera derived fromthe 250 μg rZinc vaccine of Group C; Lane 5, rHemin protein probed seraderived from the 250 μg rZinc vaccine of Group C. FIG. 7B shows aFuso-SRP Extract probed with sera derived from the combination vaccineof Group D at 10 μg Fuso-SRP Extract plus 50 μg rZinc protein. Lane 1,Fuso SRP extract probed with the sera derived from the combinationvaccine of group D. Lane 2, rZinc protein probed with sera derived fromthe combination vaccine of Group D.

FIG. 8 shows Western Blot showing the serological response to the rHeminprotein. Lane 1, Molecular Weight Marker; Lane 2, Fuso-SRP Extractprobed with sera derived from the 100 μg rHemin vaccine of Group F; Lane3, rZinc protein probed with sera derived from the 100 μg rHemin vaccineof Group E; Lane 4, rHemin protein probed with sera derived from the 25μg rHemin vaccine of Group E, and Lane 5, rHemin protein probed withsera derived from the 100 μg rHemin vaccine of Group F.

FIG. 9 shows the serological response to vaccination using therecombinant Zinc (rZinc) Protein of Fusobacterium necrophorum asanalyzed by ELISA

FIG. 10 shows the serological response to vaccination using therecombinant Hemin (rHemin) protein of Fusobacterium necrophorum asanalyzed by ELISA

FIG. 11 shows the serological response to vaccination using the Fuso-SRPExtract and the combo vaccine consisting of the rZinc protein plus theFuso SRP-Extract of Fusobacterium necrophorum as analyzed by ELISA

FIG. 12 shows SDS-PAGE gel showing the expression of the rHemin proteinat approximately 84 kDa and a hemagglutinin protein at approximately 150kDa. Lane 1, Molecular Weight Marker; Lane 2, Fuso iron-restricted andhemin supplemented SRP Extract; Lane 3, Fuso iron restricted SRPextract; Lane 4, Iron replete SRP Extract; Lane 5, Molecular weightmarker. Note that the two proteins are expressed when iron is restrictedand hemin is supplemented to the fermentation (in brackets), and not inthe presence of ferric iron or iron-restriction alone.

FIG. 13 shows Western Blots the sero-reactivity of the Fusobacteriumnecrophorum rHemin proteins and filamentous hemagglutinin 150 kDaprotein grown under iron deplete growth conditions and probed with theconvalescent calf serum of example 7 (lanes 1-4) and the rHemin mousesera as described in example 16 taken 24 hours prior to challenge (lanes5-8). Lane 1, Molecular Weight Marker; Lane 2, Fuso iron-restricted andhemin supplemented SRP Extract; Lane 3, Fuso iron restricted SRPextract; Lane 4, Iron replete SRP Extract; Lane 5, Molecular WeightMarker; Lane 6, Fuso iron-restricted and hemin supplemented SRP Extract;Lane 7, Fuso iron restricted SRP extract; Lane 8, Iron replete SRPExtract. Note the strong serologic response of the 84 kDa and 150 kDaproteins (shown in brackets in lane 2) to convalescent calf sera whengrown in iron restriction plus hemin, and the lack of serologic responsewhen the SRP extract is grown in iron limiting conditions alone, or iniron replete conditions. Also note in the brackets in lane 6 the verystrong serologic response at 84 kDa (shown in brackets) to sera frommice vaccinated with the rHemin protein. This is also not present inFuso SRP grown in iron deplete media or iron replete media as shown inlanes 7 and 8 respectively.

FIGS. 14-23, 24A-24B, 25-48, 49A-49B, and 50 show amino acid sequencesand examples of nucleotide sequences encoding the amino acid sequences.

FIG. 51A-51J shows CLUSTL Alignment of polypeptides using ClustlOmega. * (asterisk), indicates positions which have a single, fullyconserved residue.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS Polypeptides

In one aspect, this disclosure provides polypeptides and compositionsincluding polypeptides. As used herein, “polypeptide” refers to apolymer of amino acids linked by peptide bonds. Thus, for example, theterms peptide, oligopeptide, protein, and enzyme are included within thedefinition of polypeptide. This term also includes polypeptides that mayinclude one or more post-expression modifications of the polypeptidesuch as, for example, a glycosylation, an acetylation, aphosphorylation, and the like. The term polypeptide does not connote aspecific length of a polymer of amino acids. A polypeptide may beisolatable directly from a natural source or can be prepared with theaid of recombinant, enzymatic, or chemical techniques. In the case of apolypeptide that is naturally occurring, such a polypeptide is typicallyisolated.

An “isolated” polypeptide is one that has been removed from its naturalenvironment. For instance, an isolated polypeptide is a polypeptide thathas been removed from the cytoplasm or from the membrane of a cell, andmany of the polypeptides, nucleic acids, and other cellular material ofits natural environment are no longer present.

A polypeptide characterized as “isolatable” from a particular source isa polypeptide that, under appropriate conditions, is produced by theidentified source, although the polypeptide may be obtained fromalternate sources using, for example, conventional recombinant,chemical, or enzymatic techniques. Thus, characterizing a polypeptide as“isolatable” from a particular source does not imply any specific sourcefrom which the polypeptide must be obtained or any particular conditionsor processes under which the polypeptide must be obtained.

A “purified” polypeptide is one that is at least 60% free, preferably atleast 75% free, and most preferably at least 90% free from othercomponents with which they are naturally associated. Polypeptides thatare produced outside the organism in which they naturally occur, e.g.,through chemical or recombinant means, are considered to be isolated andpurified by definition, since they were never present in a naturalenvironment.

Generally, a polypeptide may be characterized by molecular weight, aminoacid sequence, nucleic acid that encodes the polypeptide, immunologicalactivity, or any combination of two or more such characteristics. Themolecular weight of a polypeptide, typically expressed in kilodaltons(kDa), can be determined using routine methods including, for instance,gel filtration, gel electrophoresis including sodium dodecyl sulfate(SDS) polyacrylamide gel electrophoresis (PAGE), capillaryelectrophoresis, mass spectrometry, liquid chromatography (includingHPLC), and calculating the molecular weight from an observed orpredicted amino acid sequence. Unless indicated otherwise, reference tomolecular weight refers to molecular weight as determined by resolving apolypeptide using an SDS polyacrylamide gel having a stacking gel ofabout 4% and a resolving gel of about 10% under reducing and denaturingconditions. A molecular weight of a protein determined by SDS-PAGE isalso referred to herein as an apparent molecular weight. In oneembodiment, the molecular weight of a protein identified by SDS-PAGEincludes molecular weights of 1, 2, 3, 4, or 5 kDa above and below thestated value.

The polypeptides described herein may be metal-regulated. As usedherein, a “metal-regulated polypeptide” is a polypeptide that isexpressed by a microbe at a greater level when the microbe is grown inlow metal conditions compared to when the same microbe is grown in highmetal conditions. Low metal and high metal conditions are describedherein. For instance, certain metal-regulated polypeptides produced byFusobacterium spp. are not expressed at detectable levels during growthof the microbe in high metal conditions but are expressed at detectablelevels during growth in low metal conditions. In one embodiment, certainmetal-regulated polypeptides produced by Fusobacterium spp. are notexpressed at detectable levels during growth of the microbe in highmetal conditions but are expressed at detectable levels during growth inlow metal conditions that also include hemin as a supplement. Table 1summarizes the expression of proteins in the absence of differentmetals.

TABLE 1 The Comparison of MW in kDA of the vaccine compositions ofFusobacterium necrophorum 1694 as Protein Analysis of examined bySDS-PAGE and MALDI-TOF-MS under various conditions of metal ionrestriction Isolate 1694 Molecular Weights in Kilodaltons (kDa) SDS-PAGEIron-deplete 103* 88 74* Iron-deplete, hemin supplemented 150 103* 88 8474* copper-deplete 126  88* 74  zinc-deplete 126 103  88 81 74*MALDI-TOF-MS Iron-deplete iron deplete, hemin supplemented 84,309copper-deplete zinc-deplete 81,723 Proteins Present in All Conditions335 243 230 220 115 SDS-PAGE Iron-deplete 68 60  52 Iron-deplete, heminsupplemented 68 60  52 copper-deplete 60* 48* 28 zinc-deplete 68 60* 4828 24 MALDI-TOF-MS Iron-deplete iron deplete, hemin supplementedcopper-deplete 48,413 zinc-deplete Proteins Present in All Conditions 4542 38 35 30 16 Protein Analysis: The molecular weights of the metalregulated proteins and porins of Fusobacterium Necrophorum were analyzedby single dimension SDS-PAGE and MALDI-TOF-MS. Note: The organism wasgrow under conditions of metal ion restriction i.e., iron-restriction;iron restriction with hemin supplementation, zinc restriction and copperrestriction. *protein is additionally enhanced under these conditions.

Examples of metal-regulated polypeptides isolatable from a Fusobacteriumspp., such as F. necrophorum, after growth in low iron conditionsinclude metal-regulated polypeptides having molecular weights of 92 kDato 79 kDa, 73 kDa to 63 kDa, 62 kDa to 58 kDa, and 57 kDa to 47 kDa.Specific examples of metal-regulated polypeptides isolatable from aFusobacterium spp., such as F. necrophorum, after growth in low ironconditions include polypeptides of 88 kDa, 68 kDa, 60 kDa, and 52 kDa.In one embodiment, the low iron condition is growth in the presence of2,2′-dipyridyl.

Examples of metal-regulated polypeptides isolatable from a Fusobacteriumspp., such as F. necrophorum, after growth in low iron conditionssupplemented with an iron-containing porphyrin, such as hemin, includemetal-regulated polypeptides having molecular weights of 155 kDa to 145kDa and 89 kDa to 79 kDa. Specific examples of this type ofmetal-regulated polypeptide isolatable from a Fusobacterium spp. aftergrowth in low iron conditions in the presence of an iron-containingporphyrin include polypeptides of 150 kDa and 84 kDa. In one embodiment,the low iron condition is growth in the presence of 2,2′-dipyridyl andhemin.

Examples of metal-regulated polypeptides isolatable from a Fusobacteriumspp., such as F. necrophorum, after growth in low copper conditionsinclude metal-regulated polypeptides having molecular weights of 131 kDato 121 kDa, 79 kDa to 69 kDa, and 33 kDa to 23 kDa. Specific examples ofmetal-regulated polypeptides isolatable from a Fusobacterium spp., suchas F. necrophorum, after growth in low copper conditions includepolypeptides of 126 kDa, 74 kDa, and 28 kDa. In one embodiment, the lowcopper condition is growth in the presence of catechin. In oneembodiment, the low copper condition is growth in the presence ofquercetin.

Examples of metal-regulated polypeptides isolatable from a Fusobacteriumspp., such as F. necrophorum, after growth in low zinc conditionsinclude metal-regulated polypeptides having molecular weights of 131 kDato 121 kDa, 108 kDa to 98 kDa, 92 kDa to 64 kDa, 53 kDa to 43 kDa, and33 kDa to 19 kDa. Specific examples of metal-regulated polypeptidesisolatable from a Fusobacterium spp., such as F. necrophorum, aftergrowth in low zinc conditions include polypeptides of 126 kDa, 103 kDa,88 kDa, 81 kDa, 68 kDa, 48 kDa, 28 kDa, and 24 kDa. In one embodiment,the low zinc condition is growth in the presence of TPEN.

In one embodiment, polypeptides described herein are expressed atdetectable levels during growth of the microbe in high metal conditionsbut more of the polypeptide is expressed during growth in low metalconditions. The expression of such polypeptides is referred to herein as“enhanced” during growth in low metal conditions. Typically, theincrease in expression of a polypeptide during growth in low metalconditions is between 20% and 500% compared to the expression of thepolypeptide during growth in high metal conditions.

Examples of metal-regulated polypeptides having enhanced expression andisolatable from F. necrophorum after growth in low iron conditionsinclude metal-regulated polypeptides having molecular weights of 108 kDato 98 kDa and 79 kDa to 69 kDa. Specific examples of metal-regulatedpolypeptides isolatable from a Fusobacterium spp., such as F.necrophorum, after growth in low iron conditions include polypeptides of103 kDa and 74 kDa.

Examples of metal-regulated polypeptides having enhanced expression andisolatable from F. necrophorum after growth in low copper conditionsinclude metal-regulated polypeptides having molecular weights of 93 kDato 83 kDa, 65 kDa to 55 kDa, and 52 kDa to 42 kDa. Specific examples ofmetal-regulated polypeptides isolatable from a Fusobacterium spp., suchas F. necrophorum, after growth in low copper conditions includepolypeptides of 88 kDa, 60 kDa, and 48 kDa.

Examples of metal-regulated polypeptides having enhanced expression andisolatable from F. necrophorum after growth in low zinc conditionsinclude metal-regulated polypeptides having molecular weights of 79 kDato 69 kDa, and 65 kDa to 55 kDa. Specific examples of metal-regulatedpolypeptides isolatable from a Fusobacterium spp., such as F.necrophorum, after growth in low zinc conditions include polypeptides of73 kDa and 60 kDa.

This disclosure also describes certain polypeptides that are notmetal-regulated. Such polypeptides are expressed in the presence of ametal ion such as, for example, in the presence of ferric chloride, andalso expressed when grown in low iron conditions. Examples of this typeof polypeptide isolatable from Fusobacterium spp., such as F.necrophorum, have molecular weights 340 kDa to 330 kDa, 247 kDa to 237kDa, 235 kDa to 215 kDa, 120 kDa to 110 kDa, 51 kDa to 25 kDa, and 21kDa to 11 kDa. Examples of molecular weights of this type of polypeptideinclude 335 kDa, 243 kDa, 230 kDa, 220 kDa, 115 kDa, 45 kDa, 42 kDa, 38kDa, 35 kDa, 30 kDa, and 16 kDa.

Other proteins provided herein include a protein at SEQ ID NO: 2, 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54 (FIGS.14-50).

In one embodiment, a polypeptide disclosed herein, for instance at SEQID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, or 54 lacks one or more amino acids from the amino terminus, e.g.,the polypeptide lacks a signal sequence. Thus, a fragment can lack atleast one, at least two, at least three, at least four, at least five,at least six, at least seven, at least eight, at least nine, at leastten, at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 30, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, at least 40, at least 41, at least 42, at least43, at least 44, at least 45, at least 46, at least 47, at least 48, atleast 49, at least 50, at least 51, at least 52, at least 53, at least54, at least 55, at least 56, at least 57, at least 58, at least 59, atleast 60, at least 61, at least 62, or at least 63 amino acids from theamino terminus of the polypeptide.

For instance, in one embodiment a polypeptide includes the followingamino acids of SEQ ID NO:2; amino acids 2 through 423, amino acids 3through 423, amino acids 4 through 423, amino acids 5 through 423, aminoacids 6 through 423, amino acids 7 through 423, amino acids 8 through423, amino acids 9 through 423, amino acids 10 through 423, amino acids11 through 423, amino acids 12 through 423, amino acids 13 through 423,amino acids 14 through 423, amino acids 15 through 423, amino acids 16through 423, amino acids 17 through 423, amino acids 18 through 423,amino acids 19 through 423, amino acids 20 through 423, amino acids 21through 423, amino acids 22 through 423, amino acids 23 through 423,amino acids 24 through 423, amino acids 25 through 423, amino acids 26through 423, amino acids 27 through 423, amino acids 28 through 423,amino acids 29 through 423, amino acids 30 through 423, amino acids 31through 423, amino acids 32 through 423, amino acids 33 through 423,amino acids 34 through 423, amino acids 35 through 423, amino acids 36through 423, amino acids 37 through 423, amino acids 38 through 423,amino acids 39 through 423, amino acids 40 through 423, amino acids 41through 423, amino acids 42 through 423, amino acids 43 through 423,amino acids 44 through 423, amino acids 45 through 423, amino acids 46through 423, amino acids 47 through 423, amino acids 48 through 423,amino acids 49 through 423, amino acids 50 through 423, amino acids 51through 423, amino acids 52 through 423, amino acids 53 through 423,amino acids 54 through 423, amino acids 55 through 423, amino acids 56through 423, amino acids 57 through 423, amino acids 58 through 423,amino acids 59 through 423, amino acids 60 through 423, amino acids 61through 423, amino acids 62 through 423, or amino acids 63 through 423.

In one embodiment, a polypeptide includes the following amino acids ofSEQ ID NO:4; amino acids 2 through 714, amino acids 3 through 714, aminoacids 4 through 714, amino acids 5 through 714, amino acids 6 through714, amino acids 7 through 714, amino acids 8 through 714, amino acids 9through 714, amino acids 10 through 714, amino acids 11 through 714,amino acids 12 through 714, amino acids 13 through 714, amino acids 14through 714, amino acids 15 through 714, amino acids 16 through 714,amino acids 17 through 714, amino acids 18 through 714, amino acids 19through 714, amino acids 20 through 714, amino acids 21 through 714,amino acids 22 through 714, amino acids 23 through 714, amino acids 24through 714, amino acids 25 through 714, amino acids 26 through 714,amino acids 27 through 714, amino acids 28 through 714, amino acids 29through 714, amino acids 30 through 714, amino acids 31 through 714,amino acids 32 through 714, amino acids 33 through 714, amino acids 34through 714, amino acids 35 through 714, amino acids 36 through 714,amino acids 37 through 714, amino acids 38 through 714, amino acids 39through 714, amino acids 40 through 714, amino acids 41 through 714,amino acids 42 through 714, amino acids 43 through 714, amino acids 44through 714, amino acids 45 through 714, amino acids 46 through 714,amino acids 47 through 714, amino acids 48 through 714, amino acids 49through 714, amino acids 50 through 714, amino acids 51 through 714,amino acids 52 through 714, amino acids 53 through 714, amino acids 54through 714, amino acids 55 through 714, amino acids 56 through 714,amino acids 57 through 714, amino acids 58 through 714, amino acids 59through 714, amino acids 60 through 714, amino acids 61 through 714,amino acids 62 through 714, or amino acids 63 through 714.

In one embodiment, a polypeptide includes the following amino acids ofSEQ ID NO:6; amino acids 2 through 736, amino acids 3 through 736, aminoacids 4 through 736, amino acids 5 through 736, amino acids 6 through736, amino acids 7 through 736, amino acids 8 through 736, amino acids 9through 736, amino acids 10 through 736, amino acids 11 through 736,amino acids 12 through 736, amino acids 13 through 736, amino acids 14through 736, amino acids 15 through 736, amino acids 16 through 736,amino acids 17 through 736, amino acids 18 through 736, amino acids 19through 736, amino acids 20 through 736, amino acids 21 through 736,amino acids 22 through 736, amino acids 23 through 736, amino acids 24through 736, amino acids 25 through 736, amino acids 26 through 736,amino acids 27 through 736, amino acids 28 through 736, amino acids 29through 736, amino acids 30 through 736, amino acids 31 through 736,amino acids 32 through 736, amino acids 33 through 736, amino acids 34through 736, amino acids 35 through 736, amino acids 36 through 736,amino acids 37 through 736, amino acids 38 through 736, amino acids 39through 736, amino acids 40 through 736, amino acids 41 through 736,amino acids 42 through 736, amino acids 43 through 736, amino acids 44through 736, amino acids 45 through 736, amino acids 46 through 736,amino acids 47 through 736, amino acids 48 through 736, amino acids 49through 736, amino acids 50 through 736, amino acids 51 through 736,amino acids 52 through 736, amino acids 53 through 736, amino acids 54through 736, amino acids 55 through 736, amino acids 56 through 736,amino acids 57 through 736, amino acids 58 through 736, amino acids 59through 736, amino acids 60 through 736, amino acids 61 through 736,amino acids 62 through 736, or amino acids 63 through 736.

In one embodiment, a polypeptide includes the following amino acids ofSEQ ID NO:34; amino acids 2 through 638, amino acids 3 through 638,amino acids 4 through 638, amino acids 5 through 638, amino acids 6through 638, amino acids 7 through 638, amino acids 8 through 638, aminoacids 9 through 638, amino acids 10 through 638, amino acids 11 through638, amino acids 12 through 638, amino acids 13 through 638, amino acids14 through 638, amino acids 15 through 638, amino acids 16 through 638,amino acids 17 through 638, amino acids 18 through 638, amino acids 19through 638, amino acids 20 through 638, amino acids 21 through 638,amino acids 22 through 638, amino acids 23 through 638, amino acids 24through 638, amino acids 25 through 638, amino acids 26 through 638,amino acids 27 through 638, amino acids 28 through 638, amino acids 29through 638, amino acids 30 through 638, amino acids 31 through 638,amino acids 32 through 638, amino acids 33 through 638, amino acids 34through 638, amino acids 35 through 638, amino acids 36 through 638,amino acids 37 through 638, amino acids 38 through 638, amino acids 39through 638, amino acids 40 through 638, amino acids 41 through 638,amino acids 42 through 638, amino acids 43 through 638, amino acids 44through 638, amino acids 45 through 638, amino acids 46 through 638,amino acids 47 through 638, amino acids 48 through 638, amino acids 49through 638, amino acids 50 through 638, amino acids 51 through 638,amino acids 52 through 638, amino acids 53 through 638, amino acids 54through 638, amino acids 55 through 638, amino acids 56 through 638,amino acids 57 through 638, amino acids 58 through 638, amino acids 59through 638, amino acids 60 through 638, amino acids 61 through 638,amino acids 62 through 638, or amino acids 63 through 638.

In one embodiment, a polypeptide includes the following amino acids ofSEQ ID NO:53; amino acids 2 through 1420, amino acids 3 through 1420,amino acids 4 through 1420, amino acids 5 through 1420, amino acids 6through 1420, amino acids 7 through 1420, amino acids 8 through 1420,amino acids 9 through 1420, amino acids 10 through 1420, amino acids 11through 1420, amino acids 12 through 1420, amino acids 13 through 1420,amino acids 14 through 1420, amino acids 15 through 1420, amino acids 16through 1420, amino acids 17 through 1420, amino acids 18 through 1420,amino acids 19 through 1420, amino acids 20 through 1420, amino acids 21through 1420, amino acids 22 through 1420, amino acids 23 through 1420,amino acids 24 through 1420, amino acids 25 through 1420, amino acids 26through 1420, amino acids 27 through 1420, amino acids 28 through 1420,amino acids 29 through 1420, amino acids 30 through 1420, amino acids 31through 1420, amino acids 32 through 1420, amino acids 33 through 1420,amino acids 34 through 1420, amino acids 35 through 1420, amino acids 36through 1420, amino acids 37 through 1420, amino acids 38 through 1420,amino acids 39 through 1420, amino acids 40 through 1420, amino acids 41through 1420, amino acids 42 through 1420, amino acids 43 through 1420,amino acids 44 through 1420, amino acids 45 through 1420, amino acids 46through 1420, amino acids 47 through 1420, amino acids 48 through 1420,amino acids 49 through 1420, amino acids 50 through 1420, amino acids 51through 1420, amino acids 52 through 1420, amino acids 53 through 1420,amino acids 54 through 1420, amino acids 55 through 1420, amino acids 56through 1420, amino acids 57 through 1420, amino acids 58 through 1420,amino acids 59 through 1420, amino acids 60 through 1420, amino acids 61through 1420, amino acids 62 through 1420, or amino acids 63 through1420.

Additional examples of polypeptides include recombinantly-producedversions of polypeptides described herein. A recombinantly-producedpolypeptide may include the entire amino acid sequence translatable froman mRNA transcript. Alternatively, a recombinantly-producedmetal-regulated polypeptide can include a fragment of the entiretranslatable amino acid sequence. For example, a recombinantly-producedmetal-regulated polypeptide may lack a cleavable sequence at eitherterminal of the polypeptide—e.g., a cleavable signal sequence at theamino terminus of the polypeptide.

Whether a polypeptide is a metal-regulated polypeptide or anon-metal-regulated polypeptide can be determined by methods useful forcomparing the presence of polypeptides, including, for example, gelfiltration, gel electrophoresis including sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillaryelectrophoresis, mass spectrometry, isobaric tags for relative andabsolute quantification (iTRAQ), and liquid chromatography includingHPLC. Separate cultures of a microbe can be grown under high metalconditions and under low metal conditions, polypeptides may be isolatedas described herein, and the polypeptides present in each culture can beresolved and compared. Typically, an equal amount of polypeptides fromeach culture is used. Preferably, the polypeptides can be resolved usingan SDS polyacrylamide gel having a stacking gel of about 4% and aresolving gel of about 10% under reducing and denaturing conditions. Forinstance, 30 micrograms (μg) of total polypeptide from each culture maybe used and loaded into wells of a gel. After running the gel andstaining the polypeptides with Coomassie Brilliant Blue, the two lanescan be compared. When determining whether a polypeptide is or is notexpressed at a detectable level, 30 μg of total polypeptide from aculture is resolved on an SDS-PAGE gel and stained with CoomassieBrilliant Blue using methods known in the art. A polypeptide that can bevisualized by eye is considered to be expressed at a detectable level,while a polypeptide that cannot be visualized by eye is considered tonot be expressed at a detectable level.

Alternatively, whether a polypeptide is a metal-regulated polypeptide ora non-metal-regulated polypeptide can be determined usingmicroarray-based gene expression analysis. Separate cultures of amicrobe can be grown under high metal conditions and under low metalconditions, RNA can be extracted from cells of each culture, anddifferences in RNA expression in cells grown in high metal conditionsversus RNA expression in cells grown in low metal conditions can bedetected and compared. For example, labeled cDNA can be prepared from8-10 μg of bacterial RNA using established protocols. The labeled cDNAcan be applied to a microarray of the Fusobacterium spp. genome. Suchmicroarrays are commercially available and evaluating gene expressionusing such arrays is routine. The polypeptides described herein may haveimmunological activity.

“Immunological activity” refers to the ability of a polypeptide toelicit an immunological response in an animal. An immunological responseto a polypeptide is the development in an animal of a cellular and/orantibody-mediated immune response to the polypeptide. Usually, animmunological response includes but is not limited to one or more of thefollowing effects: the production of antibodies, B cells, helper Tcells, suppressor T cells, and/or cytotoxic T cells, directed to anepitope or epitopes of the polypeptide. “Epitope” refers to the site onan antigen to which specific B cells and/or T cells respond so thatantibody is produced. The immunological activity may be protective.“Protective immunological activity” refers to the ability of apolypeptide to elicit an immunological response in an animal thatinhibits or limits infection by Fusobacterium spp. Whether a polypeptidehas protective immunological activity can be determined by methods knownin the art such as, for example, methods described in Examples 2 and7-11. A polypeptide may have seroactive activity. As used herein,“seroactive activity” refers to the ability of a candidate polypeptideto react with antibody present in convalescent serum from an animalinfected with a Fusobacterium spp.

A polypeptide as described herein may have the characteristics of apolypeptide expressed by a reference microbe—i.e., a referencepolypeptide. The characteristics can include, for example, molecularweight, mass fingerprint, amino acid sequence, or any combinationthereof. The reference microbe can be a gram negative, preferably amember of the family Bacteroidaceae, such as the genus Fusobacterium. Amember of the genus Fusobacterium is also referred to herein asFusobacterium spp. Examples of Fusobacterium spp. include F. necrophorum(including F. necrophorum subsp. necrophorum and F. necrophorum subsp.funduliforme), F. nucleatum, F. ulcercans, F. russi, F. varium, F.mortiferum, F. gonidiaformans, F. canifelinum; F. necrogenes; and F.naviforme. An example of a representative strain is F. necrophorum 1694.

In one embodiment, a candidate polypeptide can be considered to be apolypeptide as described herein if it has an amino acid sequence that isstructurally similar, as described in detail below, to a reference aminoacid sequence disclosed herein, for instance, SEQ ID NO: 2, 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, or a fragmentthereof, such as a fragment that lacks one or more amino acids from theamino terminus. In one embodiment, such a polypeptide is metal-regulatedwhen expressed by a Fusobacterium spp., such as F. necrophorum 1694.

As used herein, a polypeptide may be “structurally similar” to areference polypeptide if the amino acid sequence of the polypeptidepossesses a specified amount of sequence similarity and/or sequenceidentity compared to the reference polypeptide. A polypeptide also maybe “structurally similar” to a reference polypeptide if the polypeptideexhibits a mass fingerprint possessing a specified amount of identitycompared to a comparable mass fingerprint of the reference polypeptide.Thus, a polypeptide may be “structurally similar” to a referencepolypeptide if, compared to the reference polypeptide, it possesses asufficient level of amino acid sequence identity, amino acid sequencesimilarity, or a combination thereof.

Polypeptide Sequence Similarity and Polypeptide Sequence Identity

Structural similarity of two polypeptides can be determined by aligningthe residues of the two polypeptides (for example, a candidatepolypeptide and any appropriate reference polypeptide described herein)to optimize the number of identical amino acids along the lengths oftheir sequences; gaps in either or both sequences are permitted inmaking the alignment in order to optimize the number of identical aminoacids, although the amino acids in each sequence must nonetheless remainin their proper order. A reference polypeptide may be a polypeptidedescribed herein or any known metal-regulated polypeptide, asappropriate. A candidate polypeptide is the polypeptide being comparedto the reference polypeptide. A candidate polypeptide can be isolated,for example, from a microbe, or can be produced using recombinanttechniques, or chemically or enzymatically synthesized.

Unless modified as otherwise described herein, a pair-wise comparisonanalysis of amino acid sequences can be carried out using the BESTFITalgorithm in the GCG package (version 10.2, Madison Wis.).Alternatively, polypeptides may be compared using the Blastp program ofthe BLAST 2 search algorithm, as described by Tatiana et al. (FEMSMicrobiol Lett, 174:247-250 (1999)), and available on the NationalCenter for Biotechnology Information (NCBI) website. The default valuesfor all BLAST 2 search parameters may be used, includingmatrix=BLOSUM62; open gap penalty=11, extension gap penalty=1, gapx_dropoff=50, expect=10, wordsize=3, and filter on.

In the comparison of two amino acid sequences, structural similarity maybe referred to by percent “identity” or may be referred to by percent“similarity.” “Identity” refers to the presence of identical aminoacids. “Similarity” refers to the presence of not only identical aminoacids but also the presence of conservative substitutions. Aconservative substitution for an amino acid in a polypeptide may beselected from other members of the class to which the amino acidbelongs. For example, it is well-known in the art of proteinbiochemistry that an amino acid belonging to a grouping of amino acidshaving a particular size or characteristic (such as charge,hydrophobicity, or hydrophilicity) can be substituted for another aminoacid without altering the activity of a protein, particularly in regionsof the protein that are not directly associated with biologicalactivity. For example, nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and tyrosine. Polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine and glutamine. Thepositively charged (basic) amino acids include arginine, lysine andhistidine. The negatively charged (acidic) amino acids include asparticacid and glutamic acid.

Conservative substitutions include, for example, Lys for Arg and viceversa to maintain a positive charge; Glu for Asp and vice versa tomaintain a negative charge; Ser for Thr so that a free —OH ismaintained; and Gln for Asn to maintain a free —NH2. Likewise,biologically active analogs of a polypeptide containing deletions oradditions of one or more contiguous or noncontiguous amino acids that donot eliminate a functional activity—such as, for example, immunologicalactivity—of the polypeptide are also contemplated.

Thus, as used herein, reference to a polypeptide as described hereinand/or reference to the amino acid sequence of one or more SEQ ID NOscan include a polypeptide with at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% amino acidsequence similarity to the reference amino acid sequence.

Alternatively, as used herein, reference to a polypeptide as describedherein and/or reference to the amino acid sequence of one or more SEQ IDNOs can include a polypeptide with at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% aminoacid sequence identity to the reference amino acid sequence.

FIG. 51 shows cross-species sequence alignment for polypeptides havingthe amino acid sequences shown in SEQ ID NO:2, 4 and 6 (also referred toherein as proteins FT, FQ, and FN, respectively). The alignmentindicates amino acids that are conserved in the variants of eachpolypeptide across different Fusobacterium species. The alignment alsoshows regions of variability in the variants of each polypeptide acrossthe different Fusobacterium species. A person of ordinary skill in theart can deduce from such data regions of the polypeptide in whichsubstitutions, particularly conservative substitutions, may be permittedwithout unduly affecting biological activity of the modifiedpolypeptide. Further, the skilled person can use readily availablealgorithms, such as Clustl Omega, to produce alignments with relatedproteins and identify regions of conservation and variability.

Consequently, a polypeptide as described herein can include certainvariants including, for example, homologous polypeptides thatoriginate—biologically and/or recombinantly—from microbial species orstrains other than the microbial species or strain from which thepolypeptide was originally isolated and/or identified.

A polypeptide as described herein also can be designed to provide one ormore additional sequences such as, for example, the addition of codingsequences for added C-terminal and/or N-terminal amino acids that mayfacilitate purification by trapping on columns or use of antibodies.Such tags include, for example, histidine-rich tags that allowpurification of polypeptides on nickel columns. Such gene modificationtechniques and suitable additional sequences are well known in themolecular biology arts. A polypeptide as described herein also may bedesigned so that certain amino acids at the C-terminal and/or N-terminalare deleted.

A “modification” of a polypeptide as described herein includes apolypeptide (or an analog thereof such as, e.g., a fragment thereof)that is chemically or enzymatically derivatized at one or moreconstituent amino acids. Such a modification can include, for example, aside chain modification, a backbone modification, an N-terminalmodification, and/or a C-terminal modification such as, for example,acetylation, hydroxylation, methylation, amidation, and the attachmentof a carbohydrate and/or lipid moiety, a cofactor, and the like, andcombinations thereof. Modified polypeptides as described herein mayretain the biological activity—such as, for example, immunologicalactivity—of the unmodified polypeptide or may exhibit a reduced orincreased biological activity compared to the unmodified polypeptide.

A polypeptide as described herein (including a biologically activeanalog thereof and/or a modification thereof) can include a native(naturally occurring), a recombinant, a chemically synthesized, or anenzymatically synthesized polypeptide. For example, a polypeptide asdescribed herein may be prepared by isolating the polypeptide from anatural source or may be prepared recombinantly by conventional methodsincluding, for example, preparation as fusion proteins in bacteria orother host cells.

A polypeptide expressed by a reference microbe can be obtained bygrowing the reference microbe under low metal conditions as describedherein and the subsequent isolation of a polypeptide by the processesdisclosed herein. Alternatively, a polypeptide expressed by a referencemicrobe can be obtained by identifying coding regions expressed athigher levels when the microbe is grown in low metal conditions—i.e.,metal-regulated. A metal-regulated coding region can be cloned andexpressed, and the expressed metal-regulated polypeptide may beidentified by the processes described herein. A candidate polypeptidecan be isolatable from a microbe or identified from a microbe,preferably a gram negative microbe, more preferably, a member of thefamily Bacteroidaceae, such as the genus Fusobacterium., including F.necrophorum (e.g., F. necrophorum subsp. necrophorum and F. necrophorumsubsp. funduliforme), F. nucleatum, F. ulcercans, F. russi, F. varium,F. mortiferum, F. gonidiaformans, F. canifelinum; F. necrogenes; and F.naviforme.

Polynucleotide sequence similarity and polynucleotide sequence identityPolypeptides as described herein also may be identified in terms of thepolynucleotide that encodes the polypeptide. Thus, this disclosureprovides polynucleotides that encode a polypeptide as described hereinor hybridize, under standard hybridization conditions, to apolynucleotide that encodes a polypeptide as described herein, and thecomplements of such polynucleotide sequences.

As used herein, reference to a polynucleotide as described herein and/orreference to the nucleic acid sequence of one or more SEQ ID NOs caninclude polynucleotides having a sequence identity of at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to an identified reference polynucleotidesequence.

In this context, “sequence identity” refers to the identity between twopolynucleotide sequences. Sequence identity is generally determined byaligning the bases of the two polynucleotides (for example, aligning thenucleotide sequence of the candidate sequence and a nucleotide sequencethat includes, for example, a nucleotide sequence disclosed herein, suchas SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 78, or a fragment thereof) to optimize the number of identicalnucleotides along the lengths of their sequences; gaps in either or bothsequences are permitted in making the alignment in order to optimize thenumber of shared nucleotides, although the nucleotides in each sequencemust nonetheless remain in their proper order. A candidate sequence isthe sequence being compared to a known sequence—e.g., a nucleotidesequence that includes a nucleotide sequence described herein, forexample, SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 78, or a fragment thereof. For example, two polynucleotidesequences can be compared using the Blastn program of the BLAST 2 searchalgorithm, as described by Tatusova et al., (FEMS Microbiol Lett.,174:247-250 (1999)), and available on the world wide web atncbi.nlm.nih.gov/BLAST/. The default values for all BLAST 2 searchparameters may be used, including reward for match=1, penalty formismatch=−2, open gap penalty=5, extension gap penalty=2, gapx_dropoff=50, expect=10, wordsize=11, and filter on.

Finally, a polynucleotide as described herein can include anypolynucleotide that encodes a polypeptide as described herein. Thus, thenucleotide sequence of the polynucleotide may be deduced from the aminoacid sequence that is to be encoded by the polynucleotide.

This disclosure also provides whole cell preparations of a microbe,where the microbe expresses one or more of the polypeptides describedherein. The cells present in a whole cell preparation may be inactivatedsuch that the cells cannot replicate but the immunological activity ofthe polypeptides as described herein expressed by the microbe ismaintained. Typically, the cells may be killed by exposure to agentssuch as glutaraldehyde, formalin, or formaldehyde. In one embodiment,the whole cell is a member of the family Bacteroidaceae, such as thegenus Fusobacterium, including F. necrophorum (e.g., F. necrophorumsubsp. necrophorum and F. necrophorum subsp. funduliforme), F.nucleatum, F. ulcercans, F. russi, F. varium, F. mortiferum, F.gonidiaformans, F. canifelinum; F. necrogenes; and F. naviforme.

In one embodiment, a fusobacteria is engineered to express arecombinantly produced protein that has structural similarity (sequencesimilarity or sequence identity) with SEQ ID NO:2 or a fragment thereof,structural similarity (sequence similarity or sequence identity) withSEQ ID NO:4 or a fragment thereof, structural similarity (sequencesimilarity or sequence identity) with SEQ ID NO:6 or a fragment thereof,structural similarity (sequence similarity or sequence identity) withSEQ ID NO:34 or a fragment thereof, structural similarity (sequencesimilarity or sequence identity) with SEQ ID NO:53 or a fragmentthereof, or a combination thereof.

In one embodiment, a microbe, such as fusobacteria or E. coli, isengineered to express one or more recombinantly produced proteins thathave structural similarity (sequence similarity or sequence identity)with SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, or 54, or a fragment thereof.

Compositions

A composition as described herein may include at least one isolatedpolypeptide described herein, or a number of polypeptides that is aninteger greater than one (e.g., at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least 10, at least 11, at least 12, at least 13, at least14, at least 15, and so on), in any combination. Unless a specific levelof sequence similarity and/or identity is expressly indicated herein(e.g., at least 80% sequence similarity, at least 85% sequencesimilarity, at least 90% sequence identity, etc.), reference to theamino acid sequence of an identified SEQ ID NO includes variants havingthe levels of sequence similarity and/or the levels of sequence identitydescribed herein in the section headed “Polypeptide sequence similarityand polypeptide sequence identity.”

A recombinantly-produced polypeptide may be expressed from a vector thatpermits expression of the polypeptide when the vector is introduced intoan appropriate host cell. A host cell may be constructed to produce oneor more recombinantly-produced polypeptides as described herein and,therefore, can include one or more vectors that include at least onepolynucleotide encoding a polypeptide described herein. Thus, eachvector can include one or more polynucleotides as described herein—i.e.,a polynucleotide that encodes a polypeptide as described herein.Examples of host cells include, but are not limited to, E. coli andFusobacteria. Methods for the genetic manipulation of Fusobacteria areknown and routine in to art (see, for instance, Attarian et al., U.S.Pat. No. 6,962,990).

Certain compositions such as, for example, those includingrecombinantly-produced polypeptides, can include a maximum number ofdifferent types of polypeptides. In some embodiments, the maximum numberof different types of polypeptides can refer to the maximum total numberof polypeptides. Certain compositions can include, for example, no morethan 50 polypeptides such as, for example, no more than 40 polypeptides,no more than 30 polypeptides, no more than 25 polypeptides, no more than20 polypeptides, no more than 17 polypeptides, no more than 16polypeptides, no more than 15 polypeptides, no more than 14polypeptides, no more than 13 polypeptides, no more than 10polypeptides, no more than eight polypeptides, no more than sevenpolypeptides, no more than six polypeptides, no more than fivepolypeptides, no more than four polypeptides, no more than threepolypeptides, no more than two polypeptides, or no more than onepolypeptide. A non-limiting example of a composition having no more thantwo polypeptides is one having the polypeptide SEQ ID NO:2 and thepolypeptide SEQ ID NO:4. In other embodiments, a maximum number ofrecombinantly-produced polypeptides may be specified in a similarmanner. In still other embodiments, a maximum number ofnonrecombinantly-produced polypeptides may be specified in a similarmanner.

A composition can include polypeptides isolatable from one microbe, orcan be isolatable from a combination of two or more microbes. Forinstance, a composition can include polypeptides isolatable from two ormore Fusobacterium spp., or from a Fusobacterium spp. and a differentmicrobe that is not a member of the genus Fusobacterium. In certainembodiments, a composition can include a whole cell preparation in whichthe whole cell expresses one or more of the polypeptides as describedherein. In some of these embodiments, the whole cell can be aFusobacterium spp. In some embodiments, a composition can include wholecell preparations from two, three, four, five, or six strains.

In one embodiment, a composition includes at least one, at least two, atleast three, at least four, or at least five recombinantly producedproteins, for instance SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:34, and SEQ ID NO:53, or a fragment thereof. In one embodiment, acomposition includes polypeptides expressed by a Fusobacterium spp.during growth in low iron and SEQ ID NO:2 (which is not expressed at adetectable level in low iron), SEQ ID NO:4 (which is not expressed at adetectable level in low iron), SEQ ID NO:6 (which is not expressed at adetectable level in low iron when a chelator such as 2,2-dipyridyl isused to reduce the amount of available iron and is expressed at adetectable level when 2,2-dipyridyl and hemin are present), SEQ IDNO:34, SEQ ID NO:53 (which is not expressed at a detectable level in lowiron when a chelator such as 2,2-dipyridyl is used to reduce the amountof available iron and is expressed at a detectable level when2,2-dipyridyl and hemin are present), or a combination thereof. Suchcompositions are not naturally occurring. A specific example of such acomposition is one including proteins that are not detectable duringgrowth of a Fusobacterium spp., such as F. necrophorum, after growth inlow iron conditions (proteins having molecular weights of 88 kDa, 68kDa, 60 kDa, and 52 kDa), proteins having enhanced expression by aFusobacterium spp., such as F. necrophorum, after growth in low ironconditions (proteins having molecular weights of 103 kDa and 74 kDa),non-metal-regulated proteins expressed by a Fusobacterium spp., such asF. necrophorum, (335 kDa, 243 kDa, 230 kDa, 220 kDa, 115 kDa, 45 kDa, 42kDa, 38 kDa, 35 kDa, 30 kDa, and 16 kDa), and one or more recombinantlyproduced proteins selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:34, SEQ ID NO:53, or a fragment thereof. Optionally, such acomposition also includes metal-regulated proteins that are expressedafter growth in low metal conditions supplemented with hemin (150 kDaand 84 kDa).

In one embodiment, a composition includes one or more polypeptides, forinstance SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, or 54, or a fragment thereof.

Optionally, a polypeptide of the present invention can be covalentlybound to a carrier polypeptide to improve the immunological propertiesof the polypeptide. Useful carrier polypeptides are known in the art,and include, for instance, leukotoxin derived from Fusobacterium spp.The chemical coupling of a polypeptide of the present invention can becarried out using known and routine methods. For instance, varioushomobifunctional and/or heterobifunctional cross-linker reagents such asbis(sulfosuccinimidyl) suberate, bis(diazobenzidine), dimethyladipimidate, dimethyl pimelimidate, dimethyl superimidate,disuccinimidyl suberate, glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide,sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide, sulfosuccinimidyl4-(N-maleimidomethyl) cycloheane-1-carboxylate, sulfosuccinimidyl4-(p-maleimido-phenyl) butyrate and (1-ethyl-3-(dimethyl-aminopropyl)carbodiimide can be used (Harlow and Lane, Antibodies, A LaboratoryManual, generally and Chapter 5, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., N.Y. (1988)). In one embodiment, a proteindescribed herein covalently bound to a carrier protein (such as aleukotoxin) has structural similarity (sequence similarity or sequenceidentity) with SEQ ID NO:2 or a fragment thereof, has structuralsimilarity (sequence similarity or sequence identity) with SEQ ID NO:4or a fragment thereof, has structural similarity (sequence similarity orsequence identity) with SEQ ID NO:6 or a fragment thereof, hasstructural similarity (sequence similarity or sequence identity) withSEQ ID NO:34 or a fragment thereof, or has structural similarity(sequence similarity or sequence identity) with SEQ ID NO:53 or afragment thereof.

Preferably, such compositions of the present invention include lowconcentrations of lipopolysaccharide (LPS). LPS is a component of theouter membrane of most gram negative microbes (see, for instance,Nikaido and Vaara, Outer Membrane, In: Escherichia coli and Salmonellatyphimurium, Cellular and Molecular Biology, Neidhardt et al., (eds.)American Society for Microbiology, Washington, D.C., pp. 7-22 (1987),and typically includes polysaccharides (0-specific chain, the outer andinner core) and the lipid A region. The lipid A component of LPS is themost biologically active component of the LPS structure and togetherinduces a wide spectrum of pathophysiological effects in mammals. Themost dramatic effects are fever, disseminated intravascular coagulation,complement activation, hypotensive shock, and death. The non-specificimmunostimulatory activity of LPS can enhance the formation of agranuloma at the site of administration of compositions that includeLPS. Such reactions can result in undue stress on the animal by whichthe animal may back off feed or water for a period of time, andexasperate infectious conditions in the animal. In addition, theformation of a granuloma at the site of injection can increase thelikelihood of possible down grading of the carcass due to scarring orblemishes of the tissue at the injection site (see, for instance, Rae,Injection Site Reactions, available atwww.animal.ufl.edu/extension/beef/documents/SHORT94/RAE.HTM, which isavailable through the website maintained by the Department of AnimalSciences of the University of Florida, Gainesville, Fla.).

The concentration of LPS can be determined using routine methods knownin the art. Such methods typically include measurement of dye binding byLPS (see, for instance, Keler and Nowotny, Analyt. Biochem., 156, 189(1986)) or the use of a Limulus amebocyte lysate (LAL) test (see, forinstance, Endotoxins and Their Detection With the Limulus AmebocyteLystate Test, Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y.(1982)). There are four basic commercially available methods that aretypically used with an LAL test: the gel-clot test; the turbidimetric(spectrophotometric) test; the colorimetric test; and the chromogenictest. An example of a gel-clot assay is available under the tradenameE-TOXATE (Sigma Chemical Co., St. Louis, Mo.; see Sigma TechnicalBulletin No. 210), and PYROTELL (Associates of Cape Cod, Inc., EastFalmouth, Mass.). Typically, assay conditions include contacting thecomposition with a preparation containing a lysate of the circulatingamebocytes of the horseshoe crab, Limulus polyphemus. When exposed toLPS, the lysate increases in opacity as well as viscosity and may gel.About 0.1 milliliter of the composition is added to lysate. Typically,the pH of the composition is between 6 and 8, preferably, between 6.8and 7.5. The mixture of composition and lysate is incubated for 1 hourundisturbed at 37° C. After incubation, the mixture is observed todetermine if there was gelation of the mixture. Gelation indicates thepresence of endotoxin. To determine the amount of endotoxin present inthe composition, dilutions of a standardized solution of endotoxin aremade and tested at the same time that the composition is tested.Standardized solutions of endotoxin are commercially available from, forinstance, Sigma Chemical (Catalog No. 210-SE), U.S. Pharmacopeia(Rockville, Md., Catalog No. 235503), and Associates of Cape Cod, Inc.,(Catalog No. E0005). In general, when a composition of the presentinvention is prepared by isolating polypeptides from a Fusobacteriumspp. by a method as described herein (e.g., a method that includesdisrupting and solubilizing the cells, and collecting the insolublepolypeptides), the amount of LPS in a composition of the presentinvention is less than the amount of LPS present in a mixture of sameamount of the Fusobacterium spp. that has been disrupted under the sameconditions but not solubilized. Typically, the level of LPS in acomposition of the present invention is decreased by, in increasingorder of preference, at least 50%, at least 60%, at least 70%, at least80%, or at least 90% relative to the level of LPS in a compositionprepared by disrupting, but not solubilizing, the same Fusobacteriumspp.

In some aspects, a composition of the present invention does not includea leukotoxin isolatable from a Fusobacterium spp. Leukotoxins that areoptionally not present in a composition of the present invention includepolypeptides having a molecular weight of 335 kDa based on analysis withan 10% SDS-PAGE gel under reducing and denaturing conditions, and havingan activity that is toxic to bovine leukocytes (Narayanan et al.,Infect. Imun., 69, 5447-5455 (2001), and Narayanan et al., Infect.Immun., 70, 4609-4620 (2002)). Whether a polypeptide has leukotoxinactivity can be determined using the monoclonal antibody F7B10 which isreactive against a F. necrophorum leukotoxin (Tan et al., Vet.Microbiol., 42, 121-133 (1994), or by determining whether thepolypeptide is toxic to ruminant leukocytes. Methods for measuring thetoxicity of a polypeptide for ruminant leukocytes are known in the art(Narayanan et al., Infect. Imun., 69, 5447-5455 (2001), and Narayanan etal., Infect. Immun., 70, 4609-4620 (2002).

The compositions as described herein optionally further include apharmaceutically acceptable carrier. “Pharmaceutically acceptable”refers to a diluent, carrier, excipient, salt, etc., that is compatiblewith the other ingredients of the composition, and not deleterious tothe recipient thereof. Typically, the composition includes apharmaceutically acceptable carrier when the composition is used asdescribed herein. Exemplary pharmaceutically acceptable carriers includebuffer solutions and generally exclude blood products such as, forexample, whole blood and/or plasma. The compositions as described hereinmay be formulated in pharmaceutical preparations in a variety of formsadapted to the chosen route of administration, including routes suitablefor stimulating an immune response to an antigen. Thus, a composition asdescribed herein can be administered via known routes including, forexample, oral; parenteral including intradermal, transcutaneous andsubcutaneous, intramuscular, intravenous, intraperitoneal, etc. andtopically, such as, intranasal, intrapulmonary, intramammary,intravaginal, intrauterine, intradermal, transcutaneous and rectally,etc. It is foreseen that a composition can be administered to a mucosalsurface, such as by administration to the nasal or respiratory mucosa(e.g., via a spray or aerosol), in order to stimulate mucosal immunity,such as production of secretory IgA antibodies, throughout the animal'sbody.

A composition as described herein can also be administered via asustained or delayed release implant. Implants suitable for useaccording to the invention are known and include, for example, thosedisclosed in International Publication No. WO 2001/037810 and/orInternational Publication No. WO 1996/001620. Implants can be producedat sizes small enough to be administered by aerosol or spray. Implantsalso can include nanospheres and microspheres.

A composition of the present invention is administered in an amountsufficient to provide an immunological response to polypeptides or wholecells described herein. The amount of polypeptide present in acomposition can vary. For instance, the dosage of polypeptide can bebetween 0.01 micrograms (μg) and 3000 milligrams (mg), typically between10 μg and 2000 ug. When the composition is a whole cell preparation, thecells can be present at a concentration of 10⁶ bacteria/ml, 10⁷bacteria/ml, 10⁸ bacteria/ml, or 10⁹ bacteria/ml. For an injectablecomposition (e.g. subcutaneous, intramuscular, etc.) the polypeptide ispreferably present in the composition in an amount such that the totalvolume of the composition administered is 0.5 ml to 5.0 ml, typically1.0-3.0 ml. When the composition is a whole cell preparation, the cellsare preferably present in the composition in an amount that the totalvolume of the composition administered is 0.5 ml to 5.0 ml, typically1.0-2.0 ml. The amount administered will vary depending on variousfactors including, but not limited to, the specific polypeptides orcells chosen, the weight, physical condition and age of the animal, andthe route of administration. Thus, the absolute weight of thepolypeptide or number of cells included in a given unit dosage form canvary, and depends upon factors such as the species, age, weight andphysical condition of the animal, as well as the method ofadministration. Such factors can be determined by one skilled in theart. Other examples of dosages suitable for the invention are disclosedin Emery et al. (U.S. Pat. No. 6,027,736).

The formulations may be conveniently presented in unit dosage form andmay be prepared by methods well known in the art of pharmacy. Allmethods of preparing a composition including a pharmaceuticallyacceptable carrier include the step of bringing the active compound(e.g., a polypeptide or whole cell of the present invention) intoassociation with a carrier that constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active compound into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into the desired formulations.

A composition including a pharmaceutically acceptable carrier can alsoinclude an adjuvant. An “adjuvant” refers to an agent that can act in anonspecific manner to enhance an immune response to a particularantigen, thus potentially reducing the quantity of antigen necessary inany given immunizing composition, and/or the frequency of injectionnecessary in order to generate an adequate immune response to theantigen of interest. Adjuvants may include, for example, IL-1, IL-2,emulsifiers, muramyl dipeptides, dimethyldiocradecylammonium bromide(DDA), avridine, aluminum hydroxide, oils, saponins, alpha-tocopherol,polysaccharides, emulsified paraffins (available from under thetradename EMULSIGEN from MVP Laboratories, Ralston, Nebr.), ISA-70, RIBIand other substances known in the art.

In another embodiment, a composition of the invention including apharmaceutically acceptable carrier can include a biological responsemodifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha,IFN-gamma, and other cytokines that effect immune cells. A compositioncan also include an antibiotic, preservative, anti-oxidant, chelatingagent, etc. Such components are known in the art.

Methods of Making

This disclosure also provides methods for obtaining the polypeptides andwhole cells described herein. Polypeptides and whole cell preparationsdescribed herein may be obtained by incubating a member of the genusFusobacterium under conditions that promote expression of one or more ofthe polypeptides described herein. The polypeptides and whole cells asdescribed herein may be isolatable from a member of the familyBacteroidaceae, such as the genus Fusobacterium, including F.necrophorum (such as F. necrophorum subsp. necrophorum and F.necrophorum subsp. funduliforme), F. nucleatum, F. ulcercans, F. russi,F. varium, F. mortiferum, F. gonidiaformans, F. canifelinum; F.necrogenes; and F. naviforme. An example of a representative strain isF. necrophorum 1694. Microbes useful for obtaining polypeptidesdescribed herein and making whole cell preparations are commerciallyavailable from a depository such as American Type Culture Collection(ATCC). In addition, such microbes are readily obtainable by techniquesroutine and known in the art. The microbes may be derived from aninfected animal as a field isolate, and used to obtain the polypeptidesand/or the whole cell preparations as described herein, or stored forfuture use, for example, in a frozen repository at from −20° C. to −95°C., or from −40° C. to −50° C., in bacteriological media containing 20%glycerol, and other like media.

The present invention also includes compositions prepared by theprocesses disclosed herein. Typically, such conditions are low metalconditions. As used herein, the phrase “low metal conditions” refers toan environment, typically bacteriological media that contains amounts ofa free metal that cause a microbe to express a metal regulatedpolypeptide at a detectable level. As used herein, the phrase “highmetal conditions” refers to an environment that contains an amount of afree metal that causes a microbe to express a metal-regulatedpolypeptide at a decreased level compared to expression of themetal-regulated polypeptide under low metal conditions. In some cases,“high metal conditions” can refer to an environment that causes a cellto fail to express one or more of the metal-regulated polypeptidesdescribed herein at a detectable level.

In some cases, “high metal conditions” can include a metal-rich naturalenvironment and/or culture in a metal-rich medium without a metalchelator. In contrast, in some cases, “low metal conditions” can includeculture in a medium that includes a metal chelator, as described in moredetail below. Metals are those present in the periodic table underGroups 1 through 17 (IUPAC notation; also referred to as Groups I-A,II-A, IV-B, V-B, VI-B, VII-B, VIII, I-B, II-B, III-A, IV-A, V-A, VI-A,and VII-A, respectively, under CAS notation). Preferably, metals arethose in Groups 2 through 12, more preferably, Groups 3-12. Even morepreferably, the metal is iron, zinc, copper, magnesium, nickel, cobalt,manganese, molybdenum, or selenium, most preferably, iron, copper, orzinc.

Low metal conditions are generally the result of the addition of a metalchelating compound to a bacteriological medium, the use of abacteriological medium that contains low amounts of a metal, or acombination thereof. High metal conditions are generally present when achelator is not present in the medium, when a metal is added to themedium, or a combination thereof. Examples of metal chelators includenatural and synthetic compounds. Examples of natural compounds includeplant phenolic compounds, such as flavonoids. Examples of flavonoidsinclude the copper chelators catechin, naringenin, and quercetin, andthe iron chelator myricetin. Examples of synthetic copper chelatorsinclude, for instance, ammonium tetrathiomolybdate, and examples ofsynthetic zinc chelators include, for instance, N,N,N′,N′-Tetrakis(2-pyridylmethyl)-ethylene diamine (also referred to as TPEN). Examplesof synthetic iron chelators include 2,2′-dipyridyl (also referred to inthe art as α,α′-bipyridyl), 8-hydroxyquinoline,ethylenediamine-di-O-hydroxyphenylacetic acid (EDDHA), desferrioxaminemethanesulfonate (desferol), transferrin, lactoferrin, ovotransferrin,biological siderophores, such as the catecholates and hydroxamates, andcitrate.

In one embodiment, 2,2′-dipyridyl is used for the chelation of iron.Typically, 2,2′-dipyridyl is added to the media at a concentration of atleast 0.0025 micrograms/milliliter (μg/ml), at least 0.025 μg/ml, or atleast 0.25 μg/ml. High levels of 2,2′-dipyridyl can be 10 μg/ml, 20μg/ml, or 30 μg/ml.

In one embodiment, a medium is supplemented with an iron-containingporphyrin, such as hemin. Typically, hemin is added to the medium at aconcentration of 20 ug/ml, and other concentrations can be used.

In one embodiment, quercetin is used for the chelation of copper.Typically, quercetin is added to the media at a concentration of 50 uM,and concentrations between 25 μM and 100 μM can be used.

In one embodiment, TPEN is used for the chelation of zinc. Typically,TPEN is added to the media at a concentration of 50 μM is used, and itis expected that higher concentrations can be used.

It is expected that a Fusobacterium spp. with a mutation in a fur genewill result in the constitutive expression of many, if not all, of themetal regulated polypeptides of the present invention. A potential furgene has been identified in a F. nucleatum (Kapatral et al., J.Bacteriol. 184 (7), 2005-2018 (2002)). The production of a fur mutationin a Fusobacterium spp. can be produced using routine methods including,for instance, electroporation and genetic constructs useful for geneknock-out in gram negative bacteria.

In one embodiment, the fusobacteria used to make a composition describedherein, e.g., a composition including isolated polypeptides or acomposition including whole cells, may be produced using a fusobacteriathat has been engineered to recombinantly express a protein that hasstructural similarity (sequence similarity or sequence identity) withSEQ ID NO:2 or a fragment thereof, structural similarity (sequencesimilarity or sequence identity) with SEQ ID NO:4 or a fragment thereof,structural similarity (sequence similarity or sequence identity) withSEQ ID NO:6 or a fragment thereof, a portion thereof, structuralsimilarity (sequence similarity or sequence identity) with SEQ ID NO:34or a fragment thereof, structural similarity (sequence similarity orsequence identity) with SEQ ID NO:53 or a fragment thereof, or acombination thereof. In one embodiment, such a fusobacteria is incubatedin the presence of low iron conditions, and the one of more recombinantpolypeptides are expressed during the incubation in the low ironconditions. The result is a fusobacteria that expresses iron-regulatedproteins and the one of more recombinant polypeptides.

Many Fusobacterium spp. are able to grow in low metal conditions invitro in artificial media only after adaptation. For instance, aFusobacterium spp., such as the isolate given the identification numberMS 040525 and F. necrophorum 1694 can be adapted to low iron conditionsin vitro by growth in the presence of low concentrations of an ironchelator after growth in a medium containing the chelator, graduallyincreasing the concentration of the chelator. For instance, aFusobacterium spp. can be adapted to growth in low iron conditions byadding 0.0025 μg/ml of 2,2′-dipyridyl to a medium, and exposing theculture to gradually increasing concentrations of the chelator to agreater concentration, for instance 20 μg/ml as previously reportedStraub et al. (U.S. Pat. No. 8,329,192). Adaptation of Fusobacteriumspp. to reduced zinc and copper is also possible. Repeat passage of atleast five consecutive passes in 50 μM TPEN adapted Fusobacterium spp.to reduced zinc. Repeat passage of at least five consecutive passes in50 or in 100 uM quercetin, repeat passage of at least five consecutivepasses in 100 uM catechin, or repeat passage of at least fiveconsecutive passes in 100 μM Naringenin adapted Fusobacterium spp. toreduced copper. Culture of adapted Fusobacterium spp. in the presence ofany of these chelators resulted in increased expression of uniqueproteins. Adaptation of other Fusobacterium spp. strains to low metalconditions can be accomplished in this way.

The medium used to incubate the microbe is not critical, and conditionsuseful for the culture of fusobacteria are known to the skilled person.In one embodiment, supplements may be added to a culture medium, suchas, but not limited to, hemin. The volume of medium used to incubate themicrobe can vary. When a Fusobacterium spp. microbe is being evaluatedfor the ability to produce the polypeptides described herein, themicrobe can be grown in a suitable volume, for instance, 10 millilitersto 1 liter of medium. When a microbe is being grown to obtainpolypeptides for use in, for instance, administration to animals, themicrobe may be grown in a fermenter to allow the isolation of largeramounts of polypeptides. Methods for growing microbes in a fermenter areroutine and known in the art. The conditions used for growing a microbepreferably include a metal chelator, more preferably an iron chelator,for instance 2,2′-dipyridyl, TPEN, or quercetin, a pH of between 6.5 and7.5, preferably between 6.9 and 7.1, and a temperature of 37° C. When afermenter is used, the culture may be purged with an appropriate gas,for instance, nitrogen, to maintain anaerobic conditions. Members of thegenus Fusobacterium are obligate anaerobes, thus growth conditions donot include levels of oxygen that will prevent growth.

In some aspects of the invention, a Fusobacterium spp. may be harvestedafter growth. Harvesting includes concentrating the microbe into asmaller volume and suspending in a media different than the growthmedia. Methods for concentrating a microbe are routine and known in theart, and include, for example, filtration and/or centrifugation.Typically, the concentrated microbe is suspended in decreasing amountsof buffer. Preferably, the final buffer includes a metal chelator,preferably, ethylenediaminetetraacetic acid (EDTA). An example of abuffer that can be used contains Tris-base (7.3 grams/liter) and EDTA(0.9 grams/liter), at a pH of 8.5. Optionally, the final buffer alsominimizes proteolytic degradation. This can be accomplished by havingthe final buffer at a pH of greater than 8.0, preferably, at least 8.5,and/or including one or more proteinase inhibitors (e.g.,phenylmethanesulfonyl fluoride). Optionally and preferably, theconcentrated microbe is frozen at −20° C. or below until disrupted. Inone embodiment, bacterial cells may be concentrated into a pellet by,for instance, centrifugation, and the concentrated cells suspended inosmotic shock buffer (OMS; 7.26 grams/liter Tris-base and 0.93grams/liter EDTA adjusted to a pH of 8.5). The ratio of cells to OMS maybe 50 grams cell pellet, 60 grams cell pellet, or 70 grams cell pelletto 1 liter of OMS. The suspension of cells in OMS can be incubated at2-8° C. for at least 24 hours, at least 48 hours, or at least 60 hoursto remove excess endotoxin from the cells. In one embodiment, theincubation is for no greater than 72 hours. After the incubation thesuspension is centrifuged again and the supernatant discarded to removefree endotoxin and any extracellular material, e.g., secreted proteins.

When the Fusobacterium spp. is to be used as a whole cell preparation,the harvested cells may be processed using routine and known methods toinactivate the cells. Alternatively, when a Fusobacterium spp. is to beused to prepare polypeptides of the present invention, the Fusobacteriumspp. may be disrupted using chemical, physical, or mechanical methodsroutine and known in the art, including, for example, french press,sonication, or homogenization. Preferably, homogenization is used. Asused herein, “disruption” refers to the breaking up of the cell.Disruption of a microbe can be measured by methods that are routine andknown in the art, including, for instance, changes in optical density.Typically, a microbe is subjected to disruption until the percenttransmittance is increased by 20% when a 1:100 dilution is measured. Thetemperature during disruption is typically kept at 4° C., to furtherminimize proteolytic degradation.

The disrupted microbe is solubilized in a detergent, for instance, ananionic, zwitterionic, nonionic, or cationic detergent. Preferably, thedetergent is sarcosine, more preferably, sodium lauroyl sarcosinate. Asused herein, the term “solubilize” refers to dissolving cellularmaterials (e.g., polypeptides, nucleic acids, carbohydrates) into theaqueous phase of the buffer in which the microbe was disrupted, and theformation of aggregates of insoluble cellular materials. The conditionsfor solubilization preferably result in the aggregation of polypeptidesof the present invention into insoluble aggregates that are large enoughto allow easy isolation by, for instance, centrifugation.

Preferably, the sarcosine is added such that the final ratio ofsarcosine to gram weight of disrupted microbe is between 1.0 gramsarcosine per 4.5 grams pellet mass and 6.0 grams sarcosine per 4.5grams pellet mass, preferably, 4.5 gram sarcosine per 4.5 grams pelletmass. The solubilization of the microbe may be measured by methods thatare routine and known in the art, including, for instance, changes inoptical density. Typically, the solubilization is allowed to occur forat least 24 hours, more preferably, at least 48 hours, most preferably,at least 60 hours. The temperature during disruption is typically keptlow, preferably at 4° C.

The insoluble aggregates that include the polypeptides described hereinmay be isolated by methods that are routine and known in the art, suchas centrifugation, filtration, or a combination thereof. In oneembodiment, the insoluble aggregates are isolated by filtration, such astangential or crossflow filtration. Examples of a molecular weightcutoff to use with tangential filtration are 40 kDa, 50 kDa, or 60 kDa.In one embodiment, a tangential filtration system has a molecular weightcutoff of 50 kDa. Tangential filtration may aid in removal of residualsarcosine from the protein suspension. Tangential filtration results inconcentration of the protein suspension. Thus, the insoluble aggregatescan be isolated at a significantly lower cost.

In one embodiment, the sarcosine is removed from the isolatedpolypeptides. Methods for removing sarcosine from the isolatedpolypeptides are known in the art, and include, for instance,diafiltration, precipitation, hydrophobic chromatography, ion-exchangechromatography, and/or affinity chromatography, and ultrafiltration andwashing the polypeptides in alcohol, such as isopropyl alcohol, bydiafiltration. After isolation, the polypeptides suspended in buffer andstored at low temperature, for instance, −20° C. or below.

Polypeptides of the present invention may also be isolated fromFusobacterium spp. using methods that are known in the art. Theisolation of the polypeptides may be accomplished as described in, forinstance, Hussain, et al. Infect. Immun., 67, 6688-6690 (1999); Trivier,et al., FEMS Microbiol. Lett., 127, 195-199 (1995); Heinrichs, et al.,J. Bacteriol., 181, 1436-1443 (1999).

In those aspects of the present invention where a whole cell preparationis to be made, after growth of a Fusobacterium spp. the microbe can bekilled with the addition of an agent such as glutaraldehyde, formalin,or formaldehyde, at a concentration sufficient to inactivate the cellsin the culture. For instance, formalin can be added at a concentrationof 0.3% (vol:vol). After a period of time sufficient to inactivate thecells, the cells can be harvested by, for instance, diafiltration and/orcentrifugation, and washed.

In other aspects, an isolated polypeptide of the invention may beprepared recombinantly. When prepared recombinantly, a polynucleotideencoding the polypeptide may be identified and cloned into anappropriate expression host. The recombinant expression host may begrown in an appropriate medium, disrupted, and the polypeptides isolatedas described above.

Methods of Use

Also provided are methods of using the polypeptides described herein.The methods include administering to an animal an effective amount of acomposition that includes at least one polypeptide described herein. Thecomposition may further include a pharmaceutically acceptable carrier.As used herein, an “effective amount” of a composition of the presentinvention is the amount able to elicit the desired response in therecipient. The composition can be administered at a time that maternalantibody may be present, for instance, as early as one day of age, or ata later time during the life of the animal. The animal can be, forinstance, an ungulate, a companion animal, or a human. Examples ofungulates include animals that are bovine (including, for instance,cattle), caprine (including, for instance, goats), ovine (including, forinstance, sheep), porcine (including, for instance, swine), equine(including, for instance, horses), avian (including, for instance,turkeys and chickens), members of the family Cervidae (including, forinstance, deer, elk, moose, caribou and reindeer), and Bison (including,for instance, buffalo). Examples of companion animals include dogs andcats. In one embodiment, an animal is a mouse. In one embodiment, ananimal is a hooved animal. In some aspects, the methods may furtherinclude additional administrations (e.g., one or more boosteradministrations) of the composition to the animal to enhance orstimulate a secondary immune response. A booster can be administered ata time after the first administration, for instance, 1 to 8 weeks,preferably 2 to 4 weeks, after the first administration of thecomposition. Subsequent boosters can be administered one, two, three,four, or more times annually. Without intending to be limited by theory,it is expected that annual boosters will not be necessary, as an animalwill be challenged in the field by exposure to members of the genusFusobacterium expressing polypeptides having epitopes that are identicalto or structurally related to epitopes present on the polypeptidespresent in the composition administered to the animal.

In one aspect, the invention is directed to methods for making antibodyto a polypeptide described herein, for instance, by inducing theproduction of antibody in an animal, or by recombinant techniques. Theantibody produced includes antibody that specifically binds at least onepolypeptide present in the composition. In this aspect of the invention,an “effective amount” is an amount effective to result in the productionof antibody in the animal. Methods for determining whether an animal hasproduced antibodies that specifically bind polypeptides present in acomposition of the present invention can be determined as describedherein.

As used herein, an antibody that can “specifically bind” a polypeptideis an antibody that interacts only with the epitope of the antigen thatinduced the synthesis of the antibody, or interacts with a structurallyrelated epitope. An antibody that “specifically binds” to an epitopewill, under the appropriate conditions, interact with the epitope evenin the presence of a diversity of potential binding targets.

In one aspect the invention is also directed to treating an infection inan animal caused by a member of the genus Fusobacterium. The infectionmay be caused exclusively by Fusobacterium spp., or may be a mixedinfection of Fusobacterium spp. and, for instance, Bacteroides nodosus.The method includes administering an effective amount of the compositionto an animal having an infection caused by a member of the genusFusobacterium, and determining whether the Fusobacterium spp. causingthe infection has decreased. Methods for determining whether aninfection is caused by a member of the genus Fusobacterium are routineand known in the art. It is expected that compositions made withpolypeptides isolatable from one species of Fusobacterium will be usefulin the methods described herein against other species of Fusobacterium.

In another aspect, the present invention is directed to methods fortreating one or more symptoms of certain conditions in animals that maybe caused by infection by a member of the genus Fusobacterium. Examplesof conditions caused by Fusobacterium spp. infections include hepaticabscesses, foot rot, laminitis, purulent dermatitis, interdigitaldermatitis, contagious ecthyma, necrotic rhinitis, skin ulcers,peritonsillar abscesses, septic arthritis, Lemierre's syndrome,endocarditis, metritis, and shipping fever. Treatment of theseconditions can be prophylactic or, alternatively, can be initiated afterthe development of a condition described herein. Treatment that isprophylactic, for instance, initiated before a subject manifestssymptoms of a condition caused by Fusobacterium spp., is referred toherein as treatment of a subject that is “at risk” of developing thecondition. Typically, an animal “at risk” of developing a condition isan animal likely to be exposed to a Fusobacterium spp. causing thecondition. For instance, the animal is present in an area where thecondition has been diagnosed in at least one other animal, or is beingtransported to an area where a Fusobacterium spp. is endemic, and/orwhere conditions caused by Fusobacterium spp. are prevalent.Accordingly, administration of a composition can be performed before,during, or after the occurrence of the conditions described herein.Treatment initiated after the development of a condition may result indecreasing the severity of the symptoms of one of the conditions,including completely removing the symptoms. In this aspect of theinvention, an “effective amount” is an amount effective to prevent themanifestation of symptoms of a condition, decrease the severity of thesymptoms of a condition, and/or completely remove the symptoms.

The potency of a composition described herein can be tested according tostandard methods. For instance, the use of mice as an experimental modelfor Fusobacterium spp. infection in humans and large animals such ascattle is well established (Conion et al, Infect. Immun, 15, 510-517(1977), Garcia and McKay, Can. J. Comp. Med, 42, 121-127 (1978), Abe etal, Infect. Immun, 13, 1473-1478 (1976), Emery and Vaughan, Vet.Microbiol, 12, 255-268 (1986), Smith et al, Epidemiol. Infect, 110,499-506 (1993), and Narayanan et al., Vet. Micro. 93, 335-347 (2003)). Amouse model of Fusobacterium infection is available, and is recognizedas correlating to with abscess formation and useful for evaluating thein vivo efficacy of antimicrobial agents (Nagaoka et al., 2013, J. Med.Micriobiol., 62(11):1755-1759). This model has proven to be a valuablemodel to evaluate the immunogenicity and identification of varioustarget antigens provided by various fusobacteria species. Alternatively,when the condition is present in an animal such as, for instance, asheep or cow, a controlled experimental trial can be run by vaccinatinganimals with varying levels of the composition and challengingvaccinated and unvaccinated animals with a Fusobacterium spp. Methodsfor determining whether an animal has the conditions disclosed hereinand symptoms associated with the conditions are routine and known in theart. Symptoms often associated with hepatic abscesses can be a range ofpathologies, from small foci of lymphocyte inflammation surrounded bylow numbers of degenerating hepatcytes, to pronounced foci with necrosisand hemorrhage, loss of hepatocytes, fibrin and mixed inflammatory cellsat the margin of the necrotic area.

A composition of the invention can be used to provide for passiveimmunization against infection by Fusobacterium spp. For instance, thecomposition can be administered to an animal to induce the production ofimmune products, such as antibodies, which can be collected from theproducing animal and administered to another animal to provide passiveimmunity. Immune components, such as antibodies, can be collected toprepare antibody compositions from serum, plasma, blood, colostrum, etc.for passive immunization therapies. Antibody compositions includingmonoclonal antibodies, anti-idiotypes, and/or recombinant antibodies canalso be prepared using known methods. Passive antibody compositions andfragments thereof, e.g., scFv, Fab, F(ab′)2 or Fv or other modifiedforms thereof, may be administered to a recipient in the form of serum,plasma, blood, colostrum, and the like. However, the antibodies may alsobe isolated from serum, plasma, blood, colostrum, and the like, usingknown methods and spray dried or lyophilized for later use in aconcentrated or reconstituted form. Passive immunizing preparations maybe particularly advantageous for treatment of acute systemic illness, orpassive immunization of young animals that failed to receive adequatelevels of passive immunity through maternal colostrum.

Another aspect of the present invention provides methods for detectingantibody that specifically binds polypeptides of the present invention.These methods are useful in, for instance, detecting whether an animalhas antibody that specifically binds polypeptides of the presentinvention, and diagnosing whether an animal may have an infection causedby Fusobacterium spp. Preferably, such diagnostic systems are in kitform. The methods include contacting an antibody with a preparation thatincludes at least one polypeptide of the present invention to result ina mixture. Preferably, the antibody is present in a biological sample,more preferably blood, milk, or colostrum. The method further includesincubating the mixture under conditions to allow the antibody tospecifically bind a polypeptide to form a polypeptide:antibody complex.As used herein, the term “polypeptide:antibody complex” refers to thecomplex that results when an antibody specifically binds to apolypeptide. The preparation that includes the polypeptides present in acomposition of the present invention may also include reagents, forinstance a buffer, that provide conditions appropriate for the formationof the polypeptide:antibody complex. The polypeptide:antibody complex isthen detected. The detection of antibodies is known in the art and caninclude, for instance, immunofluorescence and peroxidase.

The methods for detecting the presence of antibodies that specificallybind to polypeptides of the present invention can be used in variousformats that have been used to detect antibody, includingradioimmunoassay and enzyme-linked immunosorbent assay.

The present invention also provides a kit for detecting antibody thatspecifically binds polypeptides of the present invention. The kitincludes at least one polypeptide of the present invention in a suitablepackaging material in an amount sufficient for at least one assay.Optionally, other reagents such as buffers and solutions needed topractice the invention are also included. Instructions for use of thepackaged polypeptides are also typically included.

As used herein, the phrase “packaging material” refers to one or morephysical structures used to house the contents of the kit. The packagingmaterial is constructed by known methods, preferably to provide asterile, contaminant-free environment. The packaging material has alabel which indicates that the polypeptides can be used for detectingantibodies induced by infection with Fusobacterium spp. In addition, thepackaging material contains instructions indicating how the materialswithin the kit are employed to detect such antibodies. As used herein,the term “package” refers to a solid matrix or material such as glass,plastic, paper, foil, and the like, capable of holding within fixedlimits the polypeptides. Thus, for example, a package can be amicrotiter plate well to which microgram quantities of polypeptides havebeen affixed. “Instructions for use” typically include a tangibleexpression describing the reagent concentration or at least one assaymethod parameter, such as the relative amounts of reagent and sample tobe admixed, maintenance time periods for reagent/sample admixtures,temperature, buffer conditions, and the like.

ILLUSTRATIVE EMBODIMENTS

Embodiment 1. A composition comprising:

at least one isolated polypeptide having a molecular weight of 92 kDa to79 kDa, 73 kDa to 63 kDa, 62 kDa to 58 kDa, or 57 kDa to 47 kDa, whereinthe at least one polypeptide is isolatable from a Fusobacteriumnecrophorum when incubated in media comprising an iron chelator and notisolatable when grown in the media without the iron chelator,

at least one isolated polypeptide having a molecular weight of 108 kDato 98 kDa or 79 kDa to 69 kDa, wherein the at least one polypeptide isisolatable from a Fusobacterium necrophorum when incubated in mediacomprising an iron chelator, is expressed by the Fusobacteriumnecrophorum when incubated in media without the iron chelator andexpressed at an enhanced level during growth in media comprising an ironchelator, and

at least one protein selected from the group consisting of a polypeptidehaving at least 85% similarity to SEQ ID NO:4 or a fragment thereof, apolypeptide having at least 85% similarity to SEQ ID NO:2 or a fragmentthereof, a polypeptide having at least 85% similarity to SEQ ID NO:6 ora fragment thereof, a polypeptide having at least 85% similarity to SEQID NO:34 or a fragment thereof, and a polypeptide having at least 85%similarity to SEQ ID NO:53 or a fragment thereof,

wherein the composition protects an animal against challenge withFusobacterium necrophorum.

Embodiment 2. A composition comprising:

isolated polypeptides having molecular weights of 92 kDa to 79 kDa, 73kDa to 63 kDa, 62 kDa to 58 kDa, and 57 kDa to 47 kDa, wherein thepolypeptides are isolatable from a Fusobacterium necrophorum whenincubated in media comprising an iron chelator and not isolatable whengrown in the media without the iron chelator,

isolated polypeptides having molecular weights of 108 kDa to 98 kDa and79 kDa to 69 kDa, wherein the polypeptides are isolatable from aFusobacterium necrophorum when incubated in media comprising an ironchelator, are expressed by the Fusobacterium necrophorum when incubatedin media without the iron chelator and expressed at an enhanced levelduring growth in media comprising an iron chelator, and

at least one protein selected from the group consisting of a polypeptidehaving at least 85% similarity to SEQ ID NO:4 or a fragment thereof, apolypeptide having at least 85% similarity to SEQ ID NO:2 or a fragmentthereof, a polypeptide having at least 85% similarity to SEQ ID NO:6 ora fragment thereof, a polypeptide having at least 85% similarity to SEQID NO:34 or a fragment thereof, a polypeptide having at least 85%similarity to SEQ ID NO:53 or a fragment thereof,

wherein the composition protects an animal against challenge withFusobacterium necrophorum.

Embodiment 3. A composition comprising:

isolated polypeptides having molecular weights of 92 kDa to 79 kDa, 73kDa to 63 kDa, 62 kDa to 58 kDa, and 57 kDa to 47 kDa, wherein thepolypeptides are isolatable from a Fusobacterium necrophorum whenincubated in media comprising an iron chelator and not isolatable whengrown in the media without the iron chelator,

isolated polypeptide having molecular weights of 155 kDa to 145 kDa and89 kDa to 79 kDa, wherein the at least one polypeptide is isolatablefrom a Fusobacterium necrophorum when incubated in media comprising aniron chelator and an iron-containing porphyrin and not isolatable whengrown in the media without the iron chelator and iron-containingporphyrin, and not isolatable when grown in the media with the ironchelator and in the absence of the iron-containing porphyrin, and

isolated polypeptides having molecular weights of 108 kDa to 98 kDa and79 kDa to 69 kDa, wherein the polypeptides are isolatable from aFusobacterium necrophorum when incubated in media comprising an ironchelator, are expressed by the Fusobacterium necrophorum when incubatedin media without the iron chelator and expressed at an enhanced levelduring growth in media comprising an iron chelator, and

wherein the composition protects an animal against challenge withFusobacterium necrophorum.

Embodiment 4. A composition comprising:

at least one isolated polypeptide having a molecular weight of 131 kDato 121 kDa, 79 kDa to 69 kDa, or 33 kDa to 23 kDa, wherein the at leastone polypeptide is isolatable from a Fusobacterium necrophorum whenincubated in media comprising a copper chelator and not isolatable whengrown in the media without the copper chelator, and

at least one isolated polypeptide having a molecular weight of 93 kDa to83 kDa, 65 kDa to 55 kDa, or 52 kDa to 42 kDa, wherein the at least onepolypeptide is isolatable from the Fusobacterium necrophorum whenincubated in media comprising a copper chelator, is expressed by theFusobacterium necrophorum when incubated in media without the copperchelator and expressed at an enhanced level during growth in mediacomprising an copper chelator,

wherein the composition protects an animal against challenge withFusobacterium necrophorum.

Embodiment 5. A composition comprising:

at least one isolated polypeptide having a molecular weight of 131 kDato 121 kDa, 108 kDa to 98 kDa, 92 kDa to 64 kDa, 53 kDa to 43 kDa, or 33kDa to 19 kDa, wherein the polypeptide is isolatable from aFusobacterium necrophorum when incubated in media comprising a zincchelator and not isolatable when grown in the media without the zincchelator,

at least one isolated polypeptide having a molecular weight of 79 kDa to69 kDa or 65 kDa to 55 kDa, wherein the at least one polypeptide isisolatable from the Fusobacterium necrophorum when incubated in mediacomprising a zinc chelator, is expressed by the Fusobacteriumnecrophorum when incubated in media without the zinc chelator andexpressed at an enhanced level during growth in media comprising thezinc chelator,

wherein the composition protects an animal against challenge withFusobacterium necrophorum.

Embodiment 6. A composition comprising:

isolated polypeptides having molecular weights of 131 kDa to 121 kDa, 79kDa to 69 kDa, and 33 kDa to 23 kDa, wherein the polypeptides areisolatable from a Fusobacterium necrophorum when incubated in mediacomprising a copper chelator and not isolatable when grown in the mediawithout the copper chelator, and

isolated polypeptides having molecular weights of 93 kDa to 83 kDa, 65kDa to 55 kDa, and 52 kDa to 42 kDa, wherein the polypeptides areisolatable from the Fusobacterium necrophorum when incubated in mediacomprising a copper chelator, are expressed by the Fusobacteriumnecrophorum when incubated in media without the copper chelator andexpressed at an enhanced level during growth in media comprising ancopper chelator,

wherein the composition protects an animal against challenge withFusobacterium necrophorum.

Embodiment 7. A composition comprising:

isolated polypeptides having molecular weights of 131 kDa to 121 kDa,108 kDa to 98 kDa, 92 kDa to 64 kDa, 53 kDa to 43 kDa, and 33 kDa to 19kDa, wherein the polypeptides are isolatable from a Fusobacteriumnecrophorum when incubated in media comprising a zinc chelator and notisolatable when grown in the media without the zinc chelator, and

isolated polypeptides having molecular weights of 79 kDa to 69 kDa and65 kDa to 55 kDa, wherein the polypeptides are isolatable from theFusobacterium necrophorum when incubated in media comprising a zincchelator, are expressed by the Fusobacterium necrophorum when incubatedin media without the zinc chelator and expressed at an enhanced levelduring growth in media comprising the zinc chelator,

wherein the composition protects an animal against challenge withFusobacterium necrophorum.

Embodiment 8. The composition of any one of embodiments 1-7 furthercomprising:

a polypeptide having at least 85% similarity to SEQ ID NO:4 or afragment thereof, a polypeptide having at least 85% similarity to SEQ IDNO:2 or a fragment thereof, a polypeptide having at least 85% similarityto SEQ ID NO:6 or a fragment thereof, a polypeptide having at least 85%similarity to SEQ ID NO:34 or a fragment thereof, a polypeptide havingat least 85% similarity to SEQ ID NO:53 or a fragment thereof, or acombination thereof,

wherein the composition protects an animal against challenge withFusobacterium necrophorum.

Embodiment 9. A composition comprising:

an isolated polypeptide having at least 85% similarity to SEQ ID NO:2,4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 35, 36, 37,38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, or afragment thereof, wherein the composition protects an animal againstchallenge with Fusobacterium necrophorum.

Embodiment 10. The composition of any one of embodiments 1-9 furthercomprising:

isolated polypeptides having molecular weights of 340 kDa to 330 kDa,247 kDa to 237 kDa, 247 kDa to 237 kDa, 235 kDa to 215 kDa, 120 kDa to110 kDa, 51 kDa to 25 kDa, and 21 kDa to 11 kDa, wherein thepolypeptides are isolatable from a Fusobacterium necrophorum.

Embodiment 11. The composition of any one of embodiments 1-10 furthercomprising a pharmaceutically acceptable carrier.

Embodiment 12. The composition of any one of embodiments 1-11 furthercomprising an adjuvant.

Embodiment 13. A method comprising:

administering to a subject an amount of the composition of any one ofembodiments 1-12 effective to induce the subject to produce antibodythat specifically binds to at least one polypeptide of the composition.

Embodiment 14. A method for treating an infection in a subject, themethod comprising:

administering an effective amount of the composition of any one ofembodiments 1-12 to a subject having or at risk of having an infectioncaused by a Fusobacterium spp.

Embodiment 15. A method for treating a symptom in a subject, the methodcomprising:

administering an effective amount of the composition of any one ofembodiments 1-12 to a subject having or at risk of having an infectioncaused by a Fusobacterium spp.

Embodiment 16. A method for decreasing colonization in a subject, themethod comprising:

administering an effective amount of the composition of any one ofembodiments 1-12 to a subject colonized by or at risk of being colonizedby a Fusobacterium spp.

Embodiment 17. A method for treating an infection in a subject, themethod comprising:

administering an effective amount of a composition to a subject havingor at risk of having an infection caused by a Fusobacterium spp.,wherein the composition comprises antibody that specifically binds to apolypeptide of the composition of any one of embodiments 1-12.

Embodiment 18. A method for treating a symptom in a subject comprising:

administering an effective amount of a composition to a subject havingor at risk of having an infection caused by a Fusobacterium spp.,wherein the composition comprises antibody that specifically binds to apolypeptide of the composition of any one of embodiments 1-12.

Embodiment 19. A method for decreasing colonization in a subject, themethod comprising:

administering an effective amount of a composition to a subjectcolonized by a Fusobacterium spp., wherein the composition comprisesantibody that specifically binds to a polypeptide of the composition ofembodiment any one of embodiments 1-12.

Embodiment 20. The method of any one of embodiments 13-19 wherein thesubject is a mammal.

Embodiment 21. The method of any one of embodiments 13-20 wherein themammal is a human, a bovine, or an ovine.

Embodiment 22. The method of any one of embodiments 13-21 wherein theFusobacterium spp. is F. necrophorum.

Embodiment 23. The method of any one of embodiments 13-22 wherein atleast 10 micrograms (μg) and no greater than 2000 μg of polypeptide isadministered.

Embodiment 24. The method of any one of embodiments 13-23 wherein theinfection causes a condition selected from metritis, hepatic abscesses,and foot rot.

Embodiment 25. A kit for detecting antibody that specifically binds apolypeptide, comprising in separate containers:

an isolated polypeptide of the composition of any one of embodiments1-12; and

a reagent that detects an antibody that specifically binds thepolypeptide.

Embodiment 26. A kit for detecting a polypeptide, comprising in separatecontainers:

an antibody that specifically binds an isolated polypeptide of thecomposition of any one of embodiments 1-12; and

a second reagent that specifically binds the polypeptide.

Embodiment 27. A composition comprising:

isolated antibody that specifically binds to a polypeptide of thecomposition of any one of embodiments 1-12.

Embodiment 28. A composition comprising:

an isolated whole cell that comprises a polypeptide of the compositionof any one of embodiments 1-12.

Embodiment 29. The composition of embodiment 28 wherein the isolatedwhole cell comprises the polypeptides of the composition of any one ofembodiments 1-12.

Embodiment 30. A composition comprising:

isolated antibody that specifically binds to a whole cell of any one ofembodiments 28-29.

In the preceding description, particular embodiments may be described inisolation for clarity. Unless otherwise expressly specified that thefeatures of a particular embodiment are incompatible with the featuresof another embodiment, certain embodiments can include a combination ofcompatible features described herein in connection with one or moreembodiments.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

In the following studies we examined the expression of proteins ofFusobacterium necrophorum subsp. necrophorum under various conditions ofmetal ion restriction in order to mimic the expression of novel proteinsthat may be expressed during systemic invasion and colonization of theliver.

Example 1 Selection of Fusobacterium necrophorum Isolates

More than a dozen clinical isolates of Fusobacterium necrophorum wereisolated from infected livers of beef cattle obtained from multipleprocessing plants. To preserve the original isolates a master seed stockof each isolate was prepared under anaerobic conditions. Briefly, singlecolonies were selected off blood agar plates grown under anaerobicconditions at 37° C. for approximately 48 hours. Colonies weretransferred to 10 mL modified Trypticase Soy Broth (mTSB) (BectonDickenson and Company, Franklin Lakes, N.J.) containing 0.05% cysteineHCL and 5 g/L yeast extract. A frozen master seed of each isolate wasprepared by inoculating the isolate into 10 ml mTSB. The cultures wereincubated anaerobically for 16 hours at 37° C. Five ml of each culturewas transferred into 100 ml bottles of mTSB with either 15 μg/ml2,2′-dipyridyl (DP) (Sigma Chemicals, St Louis, Mo.) or 20 μg/mL FeCl₃(Sigma) and allowed to grow anaerobically for 6 hours at 37° C. Fifty mlof the resulting cultures was combined with 50 ml of fresh mTSBcontaining 15 ug/ml DP and 20% glycerol, and the bacterial suspensionswere sterilely dispensed into 2 ml cryogenic vials (1.5 ml per vial) andstored at −80° C. until use. Master seed stocks of each isolate wereexpanded into a working seeds. Briefly, one vial of the previouslyprepared master seed was grown as previously described, sterilelydispensed into 1.5 ml per vials and stored at −80° C. until use.

One isolate was selected based on its ability to grow well in metal ionchelators and showed good outer membrane protein expression. The isolateselected as the Master for the production of the Fuso-SRP Extract wasdesignated as Master Seed 1694.

Example 2 Test for Novel Protein Expression

To test for novel protein expression under conditions of iron, zinc andcopper chelation, frozen working seeds of Fusobacterium necrophorum asdescribed above were transferred into 100 ml deplete modified TrypticSoy Broth (TSB) containing 0.05% cysteine HCL and 5 g/L yeast extractand one of four metal ion chelators, 2,2′-dipyridyl (Dp),2-pyridylmethyl-ethylene diamine (TPEN), catechin, and naringenin (allobtained from Sigma, St. Louis, Mo.). The metal ion chelators were usedat the following concentration; 50 uMN,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN); 50 and 100 uMquercetin; 100 uM catechin; or 100 uM Naringenin respectively. Cultureswere grown with each chelator for 12 hours, at which point the culturewas subcultured a second time for an additional 12 hours. Each culturewas subcultured for five consecutive passes at 12-hour intervals. At theend of the fifth pass, each culture was harvested by centrifugation at10,000×g for 20 minutes. Each culture was washed twice by centrifugationat 10,000×g and resuspended in 20 ml Tris-buffered saline, pH 7.2 at 4°C.

The bacterial cell suspensions were disrupted by sonication for 1.5minutes at 4° C. using a Branson 450 equipped with a half inchdisruption horn (Branson, Danbury Conn.). The disrupted bacterialsuspensions were clarified by centrifugation at 32,000×g for 12 minutes.The supernatants were collected and solubilized by the addition ofsodium lauroyl sarcosinate (4% vol/vol) at 4° C. for 24 hours. Thedetergent-insoluble protein-enriched fractions were collected bycentrifugation at 32,000×g for 2.5 hours at 4° C. The protein pelletswere resuspended in 200 μl Tris-buffer (pH 7.2) and stored at −90° C.

Cell extracts, derived from each metal chelation, were size-fractionatedon SDS-PAGE gels using a 4% stacking gel and 10% resolving gel. Samplesfor electrophoresis were prepared by combining 30 μg of sample with 30μl of SDS reducing sample buffer (62.5 mM Tris-HCL pH 6.8, 20% glycerol,2% SDS, 5% beta-mercaptoethanol) boiled for 4 minutes. A sample of eachextract was resolved on a 10% SDS-PAGE gel per standard methods andvisualized by Coomassie Blue staining.

The SDS-PAGE patterns of Fusobacterium necrophorum 1694 grown underiron, zinc, and/or copper chelation showed unique banding patterns thatwere different when compared to the same isolate when grown underiron-restriction in the presence of 2,2′-dyipyridyl. For example, whenthe Fusobacterium necrophorum 1694 isolate was grown underiron-restriction or in the presence of the chelator 2,2′-dyipyridyl,unique iron-regulated proteins were expressed at the 88, 84, 68, 60, and52 kDa regions (Table 1). These proteins were not detected when theisolate was grown in the presence of ferric chloride. Growth ofFusobacterium necrophorum 1694 in iron restriction also resulted in theincreased expression of proteins having molecular weights of 103 and 74kDa (Table 1). These proteins were detected when the isolate was grownin the presence of ferric chloride, but expressed at higher levelsduring growth under iron restriction.

When Fusobacterium necrophorum 1694 isolate was grown undercopper-restriction, unique copper-regulated proteins were expressed atthe 126, 74, and 28 kDa regions (Table 1). These proteins were notdetected when the isolate was grown in the presence of free copper.Growth of Fusobacterium necrophorum 1694 in copper restriction alsoresulted in the increased expression of proteins having molecularweights of 88, 60, and 48 kDa (Table 1). These proteins were detectedwhen the isolate was grown in the presence of free copper, but expressedat higher levels during growth under copper restriction.

When Fusobacterium necrophorum 1694 isolate was grown underzinc-restriction, unique zinc-regulated proteins were expressed at the126, 103, 88, 81, 68, 48, 28, and 24 kDa regions (Table 1). Theseproteins were not detected when the isolate was grown in the presence offree zinc. Growth of Fusobacterium necrophorum 1694 in zinc restrictionalso resulted in the increased expression of proteins having molecularweights of 73 and 60 kDa (Table 1). These proteins were detected whenthe isolate was grown in the presence of free zinc, but expressed athigher levels during growth under zinc restriction.

We show for the first time a novel subset of proteins expressed byFusobacterium necrophorum when the organism is grown under Iron, copperand zinc-restriction that are not expressed when the same isolate isgrown under non-restricted conditions. Since transitional metals areused by organisms to build enzymes that catalyze various biochemicalreactions, the metal ions may play a vital role in microbial survivalduring a systemic infection and/or the tissues they infect. It isperhaps for this reason that during sepsis there is a transient decreasein the availability of these transitional metals, making themunavailable for growth of the organism. These novel proteins could verywell enhance the protective efficacy of the existing composition grownunder iron-restriction because they may also be expressed by thebacteria under the metal ion restriction.

Example 3 Analysis of Proteins

The electrophoretic banding profiles of the proteins isolated from theFusobacterium necrophorum isolate grown in the following five conditionsas described above were compared: iron-deplete media (400 ml mTSBcontaining 15 μg 2,2-dipyridyl), and controlled fermentation conditions(the iron-deplete fermentation conditions of Example 4); mTSB containing100 uM naringenin; mTSB containing 100 uM catechin; mTSB containing 50or 100 uM Quercetin; and mTSB containing 100 μM of N,N,N,N Tetrakis. Theresults revealed different banding profiles between each sample grownunder different metal-depleting conditions.

In the presence of catechin (Quinde-Axtell et al., 2006, J. Agric. FoodChem., 54(26):9978-9984), a unique protein of ˜60 kDa by SDS-PAGE wasvisibly upregulated that was not enhanced in the presence of the otherflavonoids and chelators (FIG. 2; Lane 3A). This protein was furthershown to be immuno-reactive in a western blot against convalescent seraof a calf exposed to experimental challenge of Example 7 withFusobacterium necrophorum (FIG. 2; Lane 2B).

In the presence of Quercetin (copper restriction) a protein of ˜48 kDaby SDS-PAGE, was shown to be preferentially upregulated, as compared tothe other flavonoids and chelators (FIG. 1; Lane 6). This protein wasalso shown to be immuno-reactive when exposed to the convalescent serumabove (FIG. 3; Lane 2). The band from FIG. 1; Lane 6 was identified viamatrix assisted laser desorption/ionization time-of-flight spectrometry(MALDI-TOF). The closest match found via Scaffold is the outer membraneprotein of Fusobacterium necrophorum strain DAB KDE68083. The functionof this protein is unknown. The identified protein sequence was used tosearch the nucleotide sequence of F. necrophorum 1694. The nucleotidesequence and amino acid sequence identified is shown in FIG. 14 (SEQ IDNOs: 1 and 2, respectively).

A protein of ˜81 kDa by SDS-PAGE was shown to be upregulated in thepresence of N,N,N,N tetrakis, a mostly zinc chelator (FIG. 1; Lane 7 andFIG. 4 Lane A3). This protein was shown to be immuno-reactive in awestern blot against convalescent sera from an experimentally challengedcalf of Example 7 as illustrated in FIG. 4; Lane B2. The closest matchfound via Scaffold was an outer membrane protein of Fusobacteriumnecrophorum. The function of this protein is listed as TonB-dependentreceptor. The identified protein sequence was used to search thenucleotide sequence of F. necrophorum 1694. The nucleotide sequence andamino acid sequence identified is shown in FIG. 15 (SEQ ID NOs: 3 and 4,respectively).

Example 4 Production of Metal Regulated Proteins Fermentation

A cryogenic vial of the working seed of Fusobacterium necrophorum 1694(1 ml at 10⁹ CFU/ml) was used to inoculate 250 ml of 37° C. modified TSB(mTSB) containing 5 g/L yeast extract and 0.05% cysteine (Sigma) andincubated in an anaerobic chamber. The culture was incubated at 37° C.for 20 hours at which point was sterilely transferred into 1.25 litersof the above media plus 25 micrograms (μg) 2,2-dipyridyl. This secondculture was allowed to grow for an additional 3 hours at 37° C. Thisculture was used to inoculate a 15-liter Bioflo IV bench-top fermentor,(New Brunswick Scientific Co, Edison N.J.) charged with 9.5 liters ofthe above-described media. The pH was held constant between 6.9 and 7.1by automatic titration with 50% NaOH and 20% H₃PO₄. The stirring speedwas adjusted to 250 revolutions per minute (rpm), and the culture purgedwith pure nitrogen to maintain an anaerobic condition. The culture wasallowed to grow continuously at these conditions for 24 hours at whichpoint the fermentation was terminated by raising the pH to 8.5.

Harvest

The bacterial cells were concentrated by centrifugation (BeckmanCoultier, Brea, Calif.) at 7,000 rpm for 20 minutes. The bacterialpellet was then resuspended at a ratio of 60 g cell pellet to 1 litersterile Osmotic Shock Buffer (OMS) containing 7.26 grams/liter Tris-baseand 0.93 grams/liter EDTA adjusted to a pH of 8.5. The cell suspensionwas then incubated at 2-8° C. for 24 hours to remove excess endotoxinfrom the cells. The resulting suspension was then centrifuged again andthe supernate discarded to remove free endotoxin and any extracellularmaterial, e.g., secreted proteins. The cell pellet was resuspended in 3liters of OMS. The cell suspension was mixed thoroughly and dispensedinto a sterile four liter Nalgene containers and placed into a −20° C.freezer for storage. The pellet mass was calculated by centrifuging 30ml samples of the fermented culture and final harvest. Briefly,pre-weighted 50 ml Nalgene conical tubes were centrifuged at 39,000×gfor 90 minutes in a Beckman J2-21 centrifuge using a JA-21 rotor(Beckman Coulter, Brea, Calif.). At the end of the run, the supernatewas poured off and the tubes were weighed again. The pellet mass wascalculated for each stage.

Disruption (Homogenization)

One liter of the harvested three liter frozen bacterial cell slurry inOMS was thawed at 4° C. (60 gram pellet mass). The liquid culturesuspension was disrupted by homogenization. Briefly, the tank containingthe bacterial suspension was connected to a model Emulsiflex C500BHomogenizer, (Avisten Inc, Ottowa, Canada). A second process tank(empty) was connected to the homogenizer such that the fluid in theprocess tank could be passed through the homogenizer, into the emptytank and back again, allowing for multiple homogenizing passes whilestill maintaining a closed system. The temperature during homogenizationwas kept at 4° C. At the start of each pass, fluid was circulated at40-65 psi through the homogenizer and back to the tank of origin, whilethe homogenizer pressure was adjusted to ≥20,000 psi. Prior to the firstpass, two pre-homogenizing samples were withdrawn from the homogenizerto establish a baseline for determining the degree of disruption andmonitoring of pH. The degree of disruption was monitored bytransmittance (% T at 540 nanometers (nm) at 1:100 dilution) compared tothe non-homogenized sample. The bacterial suspension was passed threetimes through the homogenizer to give a final percent transmittance >80%T at a 1:100 dilution.

After homogenization, Sodium Lauroyl Sarcosinate (Hamptosyl L-30,Chem/Serv, Minneapolis, Minn.) was aseptically added to the homogenizedbacterial suspension for solubilization. The amount of Sarcosine (30%)added equaled 0.5% of the solubilizing volume, in liters. The processtank was removed from the homogenizer and kept at 4° C. while shaking at120 rpm for 16-24 hours.

Protein Harvest and Diafiltration

The protein suspension (1 Liter) was adjusted to 5 liters using sterileTris-buffer, pH 8.5. The suspension was washed and dialyzed using aOptisep 1000 SmartFlow Tangential Flow Filter device (NCSRT Inc, Apex,N.C.), equipped with a 0.8 ft² screen-channel series Alpha 50 kDaCentrasette filter (Pall Filtron) to remove residual sarcosine. Theprotein solution was concentrated by filtration to a target volume of 1liter at which point 10 liters of Tris-buffer pH 7.2 containing 10%isopropyl alcohol was slowly added to the concentrate from a secondprocess tank. Isopropyl alcohol is thought to cause a slight unfoldingof the protein structure allowing for the removal of bound sarcosinewithout compromising the immunogenicity of the proteins. Diafiltrationcontinued until the pH stabilized to 7.2 at which point 5 litersTris-buffer pH 7.2 was slowly added by diafiltration to remove residualalcohol. The Fuso-SRP Extract suspension was then concentrated toapproximately 325 ml. The protein concentrate was stored at −20° C.until use.

Alternative methods for bacterial harvest can be used. Bacterial harvestmay be performed by the use of hollow fiber filter methods. Bacterialculture is harvested using filter cartridges ranging in size from 0.2 μMto 5 kDa; preferably with a 750 kDa cartridge. Culture is reduced involume from 2-20× and subsequently washed 1-5× by diafiltration withbuffer prior to storage at 4° C. or freezing at −20° C. In this manner,undesired media proteins, bacterial proteins and LPS are removed fromthe culture. In another alternative, bacterial harvest may be performedby the use of industrial scale centrifugation, for example, by use of adisc-stack centrifuge.

Example 5 Fusobacterium Recombinant Zinc Protein (rZinc) Construction

The full nucleotide sequence of the rZinc protein, including signalpeptide, was submitted to GenScript USA Inc. (Piscataway, N.J.) for genesynthesis. The amino acid sequence was optimized for expression inEscherichia coli. Synthesized DNA was cloned in to plasmidpET-20b+(Novagen) by GenScript using the NdeI-XhoI cloning sitesallowing for a C-terminal 6× Histidine tag. The resulting plasmid isnamed rZinc_pET-20b+.

Plasmid pTHV (Epitopix, LLC) was amplified using primers 3 and 4 (Table2) to exclude the existing gene insert and only amplify the plasmidbackbone. Fragment rZinc, excluding the signal peptide, was amplifiedfrom plasmid FT_pET-20b+ using oligonucleotide primers 1 and 2 (Table2). The primers include nucleotides that overlap with the destinationplasmid, pTHV. The vector and fragment PCR products were assembled usingthe NEBuilder® HiFi DNA assembly protocol (New England Biolabs) andtransformed into NEB 10-beta competent E. coli for expression.

TABLE 2 Fusobacterium rZinc Oligonucleotide Primers Primer No. NameSequence (5′-3′) 1 FT.Fragment.FOR TCAATTTGCTAGGGGATCTGCCGAAATCGATCTGGGCAC 2 FT.Fragment.REV CCATGGCTAGCTAGCTAGTGGTGGTGGTGGTGGTGC 3 pTHV.Vector.FOR TAGCTAGCTAGCCATGGCATCAC 4pTHV.Vector.REV AGATCCCCTAGCAAATTGAA GAGAAAGATCT

Example 6 Fusobacterium Recombinant Hemin Protein (rHemin) Construction

The full nucleotide sequence of the rHemin protein, including signalpeptide, was submitted to GenScript USA Inc. (Piscataway, N.J.) for genesynthesis. The amino acid sequence was optimized for expression inEscherichia coli. Synthesized DNA was cloned in to plasmidpET-20b+(Novagen) by GenScript using the NdeI-XhoI cloning sitesallowing for a C-terminal 6× Histidine tag. The resulting plasmid isnamed rHemin_pET-20b+.

Plasmid pTHV (Epitopix, LLC) was amplified using primers 7 and 8 (Table3) to exclude the existing gene insert and only amplify the plasmidbackbone. Fragment rHemin, excluding the signal peptide, was amplifiedfrom plasmid rHemin_pET-20b+ using oligonucleotide primers 5 and 6(Table 3). The primers include nucleotides that overlap with thedestination plasmid, pTHV. The vector and fragment PCR products wereassembled using the NEBuilder® HiFi DNA assembly protocol (New EnglandBiolabs) and transformed into NEB 10-beta competent E. coli forexpression.

TABLE 3 Fusobacterium rHemin Oligonucleotide Primers Primer No. NameSequence (5′-3′) 5 FH.Fragment.FOR TACTGTTATAGATCTTTCTGAACAAACGATTGAACTGGG 6 FH.Fragment.REV TCCCTGCCTCTGTCACTTCCTTTCGGGCTTTGTTAG 7 pTHV.201601.FOR TGACAGAGGCAGGGAGTG 8pTHV.201601.REV AGAAAGATCTATAACAGT AGCCATATTTAAAC

Example 7 Preparation of Convalescent Sera in Holstein Calves

Convalescent serum was collected as part of a vaccination and challengestudy in which steers with an average weight of approximately 350 poundswere used for generation of sera. Calf number 72 was an unvaccinatedcontrol animal challenged via the portal vein according to the method ofK. Lechtenberg et al (Am J Vet Res. 1991 June; 52(6)803-9) withapproximately 6×10⁸ cfu of a virulent Fusobacterium necrophorum strain.

Example 8 Blood Sample Collection

Blood samples were collected from all steers on day 66 (10 dayspost-challenge). All blood was collected in sterile 13×75 millimetervacutainer collection tubes (SST No. 369783, Becton Dickinson, FranklinLakes, N.J.). After clotting the blood tubes were centrifuged at 800×gfor thirty minutes and frozen at −20° C.

Example 9 Identification of Sero-Reactive Membrane Proteins ofFusobacterium necrophorum Using Western Blot Analysis

The proteins in the vaccine composition as described in Example 2 weresubjected to electrophoresis followed by western blot analysis withconvalescent serum as described in Example 7. Briefly, the membraneproteins derived from Fusobacterium necrophorum grown underiron-limiting conditions were size-fractionated on an SDS-PAGE gel usinga 4% stacking gel and 7.5% resolving gel. A 10 μl sample was combinedwith 10 μl of SDS reducing sample buffer (62.5 mM Tris-HCL ph 6.8, 20%glycerol, 2% SDS, 5% β-mercaptoethanol) and boiled for 4 minutes.Samples were electrophoresed at 18 mA constant current for 5 hour at 4°C. using a Protein II xi cell and model 1000/500 power supply (BioRadLaboratories, Richmond, Calif.). Band migration was visualized usingbroad range kaleidoscope standards (BioRad) to aid in the electro-blottransfer while biotinylated broad range standards were used as molecularweight references on the blot, see FIG. 7. For Western blot analysis,proteins were electroblotted from the gel onto trans-blot nitrocellulosemembranes (BioRad) overnight, at 4° C. at 50 V, in Towbin buffer (25 mMTris, 192 mM glycine, 20% methanol) using a BioRad Trans-Blot transfercell and a Pac 300 power supply (BioRad). The nitrocellulose membranewas blocked using 3% fish gelatin (Sigma Chemical, St. Louis, Mo.) inTris buffered saline (TBS-20 mM Tris, 500 mM NaCl, pH 7.5) for 1 hourwhile shaking at 37° C. The membrane was dried at 37° C. and blocked inTBS containing 3% fish gelatin and this process was repeated. Themembrane was then probed with the polyclonal convalescent sera collectedfrom the challenged steer as described in example 7. The primaryantibody was diluted 1/500 in TBS containing 1% fish gelatin, 0.05%Tween 20 and 0.2% sodium azide (Antibody Buffer). The membrane wasincubated with the primary antibody solution overnight on a shaker atroom temperature. The membrane was then washed two times in TBScontaining 0.05% Tween 20 (TTBS) and transferred to antibody buffercontaining a 1/10,000 dilution of Alkaline phosphatase-conjugated mouseanti-bovine IgG clone BG-18 (Sigma) and a 1/3000 dilution of avidinconjugated to alkaline phosphatase (BioRad). The membrane was incubatedat 37° C. for 2 hours on a shaker, then washed in TTBS four times toremove unbound conjugate. The blot was resolved in substrate solutioncontaining alkaline phosphate color reagent A and B in 1×AP colordevelopment Buffer (BioRad) for 30 min. at 37° C. on a shaker. Theresulting Western immunoblot was documented using a BioRad GS-800Densitometer (see FIGS. 2-4, 7 and 8).

The purpose of this analysis was to determine which of the proteinspresent in the immunizing composition induced antibody responsesfollowing challenge of steers. The results revealed unique immunologicalreactivity with proteins at 48 kDa in the presence of the copperchelator Quercetin, catechin, or narangenin (FIGS. 2, 3 and 4); at ˜60kDa in the presence of the copper chelator catechin (FIG. 2); and an ˜82kDa protein in the presence of the zinc chelator Tetrakis (TPEN) (FIG.4); and an ˜90 kDa protein in the presence of quercetin. In addition,the results revealed unique immunological reactivity proteins at 131kDa, 85 kDa, 60 kDa, and in the area of 40-43 kDa in the presence of thecopper chelator Quercetin; at 107 kDa, 75 kDa, 60 kDa, and in the areaof 40-43 kDa in the presence of the copper chelator catechin; at 73 kDaand in the area of 40-43 kDa in the presence of the copper chelatornaringenin; and at 82 kDa, 75 kDa, 73 kDa, 60 kDa, 48 kDa, and in thearea of 40-43 kDa in the presence of the zinc chelator Tetrakis (TPEN).The molecular weights of the immunologically reactive proteins are notidentical with the molecular weights of the metal regulated proteinsdescribed herein identified by SDS-PAGE; however, the molecular weightsof the immunologically reactive were determined using the results ofwestern immunoblot assays, and the skilled person will recognize thatthe ability to accurately determine molecular weights from a westernimmunoblot is reduced.

These results demonstrated that the membrane proteins of the compositiondescribed in Example 2 reacted strongly with the convalescent seradescribed in Example 7, suggesting that these components of the vaccinemay provide protection against disease. However, the sensitivity limitsof the assay may have prevented the detection of weaker interactions,that, although less evident, may still contribute to the vaccine'seffectiveness by augmenting the immune response to the composition. Inaddition, the proteins that were not sero-reactive in this assay mayelicit responses other than antibody production, such as stimulation ofcytokines, intereferon, interleukins, T-cells, or colony-stimulatingfactors. Such responses could enhance, direct, or restore the ability ofthe host's immune system to fight disease.

Example 10 Preparation of the Immunizing Compositions Derived fromFusobacterium necrophorum

The composition made from Fusobacterium necrophorum strain 1694 ofExample 4 was used as the vaccine in this experimental study. Thevaccine was prepared from the composition by diluting the antigen intophosphate buffered saline (PBS) containing 8.0 NaCl, 0.2 KCl, 1.44 g/lNa₂HPO₄ and 0.24 g/l KH₂PO₄ pH 7.4 The suspension (500 μg totalprotein/ml) was then emulsified into the commercial adjuvant, EMULSIGEN,(MVP Laboratories, Ralston, Nebr.) using the syringe method ofemulsification. The process can be summarized as follows: (1) force anamount of adjuvant from syringe B by pushing it into syringe A filledwith antigen solution to mingle with the latter; (2) push the samevolume of the mix from syringe A back to syringe B slowly; (3) repeatthe above mixing process until the mixed portion becomes milky white. Amouse dose was administered to give a final dose of 100 μg total proteinin a 0.1 ml injectable volume with an adjuvant concentration of 22.5%vol/vol. A placebo was prepared by replacing the antigen withphysiological saline in the above formulation and emulsifying thesuspension into EMULSIGEN to give an adjuvant concentration of 22.5%.

Example 11 Mouse Vaccination

The efficacy of the Fuso-SRP Extract derived from Fusobacteriumnecrophorum 1694 was carried out against a live virulent challenge inmice. Eighty (N=80) female CF-1 mice obtained from Charles RiverLaboratories (Wilmington Del.) weighing 16-22 grams were equallydistributed into two groups (40 mice/group). Mice were housed inpolycarbonate cages in a self-contained HEPA filtered Mobile HousingSystem (Thoren Caging systems; Hazleton; PA). Treatment groups weredesignated as Group-A (Placebo) and Group-B (Fuso-SRP ExtractVaccinated). Food and water was supplied ad libitum to all mice. Micewere vaccinated subcutaneously twice at 21 day intervals. The volumeadministered was 0.1 ml/mouse see Table 4.

TABLE 4 Experimental Design Vaccine # Vac- Total Volume Vac- cine GroupsMice Vaccine Antigen Adjuvant (ml) cines Route A 40 Placebo N/A 22.5%0.1 2 SQ Emulsigen B 40 Fuso-SRP 100 μg 22.5% 0.1 2 SQ Extract Emulsigen

Example 12 Preparation of Challenge Organism

Twenty eight days after the second vaccination, mice in groups A and Bwere intravenously challenged. The Fusobacterium necrophorum isolate1694 as previously described in Example 1 was used as the challengestrain. Briefly, a cryogenic vial of the frozen working seed ofFusobacterium necrophorum 1694 of Example 1 was used for challenge.Briefly, the frozen stock was thawed at 4° C. then diluted 1:10 in coldmTSB and the resulting dilution was used for challenge. All mice ingroups A and B were intravenously challenged via the caudal vein with0.1 ml of Fusobacterium necrophorum (˜1×10⁸ colony forming units per ml)as previously enumerated as described in Example 7 Just prior tochallenge, 100 μl of the above bacterial suspension was serially dilutedtenfold to enumerate the number of CFU/dose. Mortality was recordeddaily for 10 days post challenge at which point the experimental trialwas terminated. All surviving mice from Groups A- and B were euthanizedby carbon dioxide. The liver from all dead and surviving mice wasaseptically removed and gross examination was performed to determinedifferences in liver abscessation.

Example 13 Challenge Results

The results showed a strong protective index against a caudal veinchallenge as seen in Table 5. Ten out of 40 (25%) of theplacebo-vaccinated mice (Group A) died within 10 days after challenge.In contrast, no mortality (0 out of 40) was seen in the vaccinated miceof Group B (degree of significance of P=0.001).

TABLE 5 Comparison of Mortality; Liver Abscess and Percent Survivabilitybetween Vaccinated and Placebo Controls Following Intravenous Challengewith Fusobacterium necrophorum ^(b)Liver ^(c)Percent Groups Mice^(a)Mortality (%) Lesions (%) Survivability A) Placebo 40 10 (25) 9(22.5) 75 B) Fuso-SRP 40 0 1 (2.5)  100 Extract ^(a)The mortality ofmice that died within 10 days after IV challenge with 3.0 × 10⁸ CFU ofFusobacterium necrophorum. ^(b)The percent of mice that had visibleliver abscess upon death or at 10 days post challenge (two-sided Pvalue) was P = 0.0143. ^(c)Percent Survivability; 100 percent of thevaccinated mice survived challenge compared to the non-vaccinatedcontrols where only 75 percent survived (two-sided P value) was P =0.0010.

Gross examination of each liver revealed a dramatic difference in thenumber of abscesses between the Placebo and Vaccinated mice. It wasclearly evident that mice given the vaccine rapidly reduced the numberof bacteria able to proliferate successfully in the liver as indicatedby the reduction in visible abscesses as compared to the placebovaccinated mice (Table 5). The difference in the number of abscessedlivers of Placebo vaccinated controls and the vaccinated group wasstatistically significant (degree of significance of P=0.0143),indicating a direct correlation in the reduction of lesions throughvaccination by preventing the proliferation and colonization ofFusobacterium necrophorum in the liver The number of mice with abscesseswas 9 out of 40 (22.5%) in the placebo vaccinated group as compared toonly 1 out of 40 (2.5%) in the vaccinated group (Table 5).

The Fuso-SRP Extract vaccine of Group B showed a high degree of systemicprotection as compared to non-vaccinated mice of Group A; (Placebovaccinated). The vaccine prepared from Fusobacterium necrophorum washighly efficacious in preventing mortality associated with anintravenous challenge with Fusobacterium necrophorum in a standardizedmouse model as well as reducing the formation of liver abscesses.

Example 14 Vaccine-Mediated Protection of Novel Recombinant Zinc andHemin Proteins of Fusobacterium necrophorum in a Mouse Sepsis Model

The purpose of the following experimental study was to evaluate thevaccine efficacy of two recombinant proteins, rZinc and rHemin ofFusobacterium necrophorum. In addition, a vaccine formulation consistingof the rZinc protein in combination with the Fuso-SRP extract and theFuso-SRP extract as a stand-alone vaccine formulation was evaluated asillustrated in Table 6. The bovine strain of Fusobacterium necrophorum1694 was used as the challenge strain as previously described inExample 1. The outcome parameters used to evaluate vaccine efficacy inthis experiment were 1) serological response to vaccination 1) thereduction in the incidence of lesions between vaccinates and placebocontrol mice 2) the difference in the size of lesions based on a lesionscore, where a lesion ≤0.5 cm=1 and a lesion ≥0.5=2) the difference inthe Prevented Fraction which is defined as the percentage of animals ineach treatment group that is protected against liver lesions calculatedas:

1−p₂/p₁

p₂=affected fraction in vaccine group

p₁=affected fraction in control group

where, the prevented fraction is the complement of the risk ratio1−p₂/p₁; where p₂ is the affected fraction in the experimental productand p₁ is the affected fraction in the placebo group. The precision ofthe estimate is evaluated by determining the 95% confidence interval.

Briefly, three hundred twenty (N=320) female Harlan CF-1 mice obtainedfrom Charles River Laboratory (Wilmington, Mass.) weighing 16-22 gramswere equally divided into 8 treatment groups (40 mice/group) designatedas groups A-H (Table 6). Mice were housed in polycarbonate cages in aself-contained HEPA filtered Mobile Housing System (Thoren Cagingsystems; Hazleton; PA) at 5 mice per cage with food and water suppliedad libitum. All mice were allowed to acclimate one week prior to thefirst vaccination.

Example 15 Vaccine Preparation and Vaccination

Vaccines of the recombinant Zinc and Hemin proteins as well as theFusobacterium necrophorum SRP extract was prepared at their appropriatedosage levels in phosphate buffered saline (PBS) containing 8.0 g/lNaCl, 0.2 g/l KCl, 1.44 g/l Na.sub.2HPO.sub.4 and 0.24 g/lKH.sub.2PO.sub.4 pH 7.4 formulated with 10 percent Rehydragel HPA;(General Chemical; Berkeley Heights; New Jersey). The antigen/aluminumhydroxide suspensions was stirred for 24 hours at 4° C. to allow maximumadsorption of the protein to the adjuvant. The antigen/aluminumhydroxide suspension was then emulsified into the commercial adjuvant,EMULSIGEN, (MVP Laboratories, Ralston, Nebr.) to give and adjuvantconcentration of 22.5% vol/vol. The rZinc vaccine of groups B and C wasformulated at 100 μg and 250 μg respectively. The combination vaccine ofGroup D was formulated containing 10 μg of the Fuso-SRP extract aspreviously described in Example 4 and 50 μg of the rZinc protein to givea mouse dose of 60 μg total protein. The rHemin protein of Groups E andF was formulated at 25 μg and 100 μg dose levels respectively, while theFuso-SRP extract of Groups G and H was formulated at 10 and 100 μg totalprotein respectively. All vaccines of Groups A-H were formulated to bedelivered at 0.1 ml injectable volume. A placebo vaccine was prepared bysubstituting physiological saline for the aqueous protein suspension asdescribed above. Mice were vaccinated subcutaneously two times at 21 dayintervals and then challenged 14 days following the last vaccination.Blood was taken randomly from five mice from each group three timesduring the course of the study 1) first vaccination (pre-immune); 2)second vaccination and 3) 24 hours pre-challenge. Individual bloodsamples were equally pooled and stored at −80° C. until analyzed bywestern blot and ELISA to determine the serological response tovaccination.

TABLE 6 Experimental Design Total Vaccine Volume # Vaccine Group MiceVaccine Antigen Adjuvant (ul) Vaccines Route A 40 Placebo N/A 10% ALOH +N/A 2 SQ 22.5% Emulsigen *B  40 rZinc 100 μg 10% ALOH + 100 1 SQ 22.5%Emulsigen C 40 rZinc 250 μg 10% ALOH + 100 2 SQ 22.5% Emulsigen D 40rZinc + Fuso- 10 μg SRP + 10% ALOH + 100 2 SQ SRP Extract  50 μg 22.5%rZinc Emulsigen E 40 rHemin  25 μg 10% ALOH + 100 2 SQ 22.5% Emulsigen F40 rHemin 100 μg 10% ALOH + 100 2 SQ 22.5% Emulsigen G 40 Fuso-SRP  10μg 10% ALOH + 100 2 SQ Extract 22.5% Emulsigen H 40 Fuso-SRP 100 μg 10%ALOH + 100 2 SQ Extract 22.5% Emulsigen Please note; the recombinantzinc protein (*B) in the above experimental design was inadvertentlyvaccinated only one time rather than the proposed two time vaccinationregimen.

Example 16 Preparation of Challenge Organism

The Fusobacterium necrophorum isolate 1694 as previously described inExample 1 was used as the challenge strain. Briefly, a cryogenic vial ofthe frozen working seed of Fusobacterium necrophorum 1694 of Example 1was used for challenge. The frozen stock was thawed at 4° C. thendiluted 1:10 in cold mTSB and the resulting dilution was used forchallenge. All mice in groups A through H were intravenously challengedvia the caudal vein with 0.1 ml of Fusobacterium necrophorum (˜1×10⁸colony forming units per ml) as previously enumerated as described inExample 7. Just prior to challenge, 100 μl of the above bacterialsuspension was serially diluted tenfold to enumerate the number ofCFU/dose. Mortality was recorded daily for 7 days post challenge atwhich point the experimental trial was terminated. All surviving micefrom Groups A-H were euthanized by carbon dioxide. The liver from alldead and surviving mice was aseptically removed and gross examinationwas done to determine differences in liver abscessation.

Example 17 Challenge Results

Seven days post challenge the livers of all dead and surviving mice wereaseptically removed and the difference in incidence and size of lesionswas determined between vaccinates and placebo controls. Grossexamination of each liver revealed a dramatic difference in both thesize and incidence of lesions between the Placebo and Vaccinated mice.For example; thirty percent of the Placebo control mice had well definedfoci in the livers in contrast to vaccinates; (Table 7; FIG. 5). Boththe rZinc and rHemin proteins showed a reduction in the incidence oflesions to 10 and 15 percent respectively at the 250 μg (rZinc) and 25μg (rHemin) dose level compared to controls which showed an incidencerate of 30 percent, see Table 7; FIG. 5). It is interesting note thedifference in the vaccine dose between the two recombinant proteins thatinduced efficacy i.e., 250 μg for the rZinc protein and 25 μg for therHemin protein of Groups C and E (Table 7; FIG. 5). Both vaccineformulations of the Fuso-SRP Extracts of Groups G and H at the 10 μg and100 μg dose level were highly effective at reducing the incidence oflesions compared to the Placebo control of Group A. In fact; the vaccineat 10 μg dose level completely protected mice from abscessation and only1 out of 40 mice in the 100 μg dose level of Group H showed lesions inthe liver. In comparison; the combo vaccine of Group D containing 10 μgof the Fuso-SRP extract and 50 μg of the rZinc protein was also highlyeffective in reducing the incidence of lesions; only 3 percent or 1 outof 40 mice were found to have lesions.

TABLE 7 The percent difference of lesions in the liver and thecalculated Prevented Fraction between vaccinates compared to the placebocontrol ^(a)Liver ^(b)Prevented Treatment Total Mortal- Lesions FractionGroups (A-H) Antigen ity (%) (%) A) Placebo (N = 40) N/A 4 30 0 B) rZinc100 μg 2 30 0 C) rZinc 250 μg 1 10 73 D) rZinc + Fuso- 10 μg SRP + 0 392 SRP Extract 50 μg rZinc E) rHemin  25 μg 3 13 58 F) rHemin 100 μg 015 50 G) Fuso-SRP Extract  10 μg 1 0 100 H) Fuso-SRP Extract 100 μg 0 392 ^(a)Liver lesions - The difference in the number of mice havinglesions calculated as a percent between treatment groups compared tocontrols. ^(b)Prevented fraction is the percentage of mice in eachtreatment group that was protected against liver lesions.

FIG. 6 shows the difference in the size of lesions between vaccinatesand controls. Please note; the significant reduction in the size of thelesions in all vaccinated groups (C—H) with the greatest reduction beingin the Fuso-SRP Extract formulations at both the 10 and 100 μg doselevels. As illustrated; it is clearly evident that each vaccineformulation including the recombinant rZinc; rHemin and ExtractedFuso-SRP proteins reduced the number of bacteria able to proliferatesuccessfully in the liver as indicated by the reduction in visibleabscesses as well as the size of lesions compared to the placebovaccinated mice; see FIGS. 5 and 6.

The only vaccinated group that was not significantly different than theplacebo controls was the rZinc protein at the 100 μg dose level of GroupB. This vaccine was inadvertently administered only one time rather thanthe proposed two time vaccine regimen (Table 6). These results clearlyshow that a single dose of the rZinc protein at a 100 μg is notsufficient to induce a proper protective response. The rZinc proteinadministered at the 250 μg dose level of Group C was highly effective inreducing both the incidence and the size of lesions, clearlydemonstrating a dose response; as the dose increased the incidence andsize of lesions decreased. It's interesting to speculate that if thedose of the rZinc protein was increased beyond the 250 μg dose level ifone could have obtained a greater degree of protection that would havebeen equivalent to the Fuso-SRP Extract. These results clearlydemonstrate that a single recombinant protein at an optimal dose canprotect against a systemic challenge of Fusobacterium. The rZinc proteinreduced the incidence and the size of lesions.

Not unlike the rZinc protein, the rHemin protein was also effective as avaccine candidate in reducing both the incidence and the size of lesionscompared to the non-vaccinated controls. Both the 25 μg and 100 μg doselevels of the rHemin protein reduced the incidence and overall size oflesions in the liver (FIGS. 5 and 6). Due to the lack of availability offinal antigen of this protein the experiment did not allow for dosematching of the two recombinant proteins; i.e., it would have been moreappropriate to compare the recombinant proteins at the same dose levelsrather than at different protein amounts. Nevertheless, results clearlydemonstrate that the rHemin protein is an excellent target antigen forcontrolling both the size and incident of liver lesions.

TABLE 8 The percentage of animals in each treatment group that isprotected against liver lesions *PREVENTED P VALUE BY TREATMENT GROUPSFRACTION FISHER'S ZINC 100 UG SINGLE DOSE  0% ZINC 250 UG 73% 0.048 ZINC50 UG/SRP 10 UG 92% 0.0015 HEMIN 25 UG 58% 0.099 HEMIN 100 UG 50% 0.18SRP 10 UG 100%  0.0002 SRP 100 UG 92% 0.0015 *Prevented Fraction isdefined as the percentage of animals in each treatment group that isprotected against liver lesions calculated as (1 − p₂/p₁) where p₂ isthe affected fraction in the vaccine groups and p₁ is the affectedfraction in placebo control group.

In addition, the results show the calculated Prevented Fraction asdescribed in Example 14 for each treatment group compared to thenon-vaccinated placebo controls. For example, the Fuso-SRP Extracts atboth the 10 μg and 100 μg dose levels had calculated Prevented Fractionsof 100 and 92 percent with p-values of 0.0002 and 0.0015 respectively(Table 8). In fact the only other group that equaled these values wasthe combo vaccine of Group D consisting of 50 μg of the rZinc proteinplus 10 μg of the Fuso-SRP Extract having a Prevented Fraction of 92percent. These results showed a high degree of statistical significancehaving a p-value of 0.0015. The rZinc at the 250 μg dose level had aPrevented Fraction of 73 percent with a degree of significance ofp=0.048. The rHemin protein at the 25 μg and 100 μg dose levels hadPrevented Fractions of 58 and 50 percent with degrees of significance ofp=0.099 and p=0.180 respectively. The rHemin protein showed a reductionin the incidence and the size of lesions when compared to thenon-vaccinated controls but was not statistically significant. Resultsmay have been different if a more rigorous dose finding regiment wouldhave been performed. Nevertheless, all vaccine formulations testedexcept for Group B showed a reduction in the incidence and the overallsize of liver lesions.

Example 18 Western Blot

First the rZinc; rHemin and Fuso-SRP Extract were subjected toelectrophoresis followed by western blot analysis with the sera taken 24hours pre-challenge as described in Example 15. Briefly, rZinc; rHeminand Fuso-SRP Extract were size-fractionated on an SDS-PAGE gel using a4% stacking gel and 7.5% resolving gel. A 10 μl sample was combined with10 μl of SDS reducing sample buffer (62.5 mM Tris-HCL ph 6.8, 20%glycerol, 2% SDS, 5% β-mercaptoethanol) and boiled for 4 minutes.Samples were electrophoresed at 18 mA constant current for 5 hour at 4°C. using a Protein II xi cell and model 1000/500 power supply (BioRadLaboratories, Richmond, Calif.). Band migration was visualized usingbroad range kaleidoscope standards (BioRad) to aid in the electro-blottransfer while biotinylated broad range standards were used as molecularweight references on the blot, see FIGS. 7 and 8. For Western blotanalysis, proteins were electroblotted from the gel onto trans-blotnitrocellulose membranes (BioRad) overnight, at 4° C. at 50 V, in Towbinbuffer (25 mM Tris, 192 mM glycine, 20% methanol) using a BioRadTrans-Blot transfer cell and a Pac 300 power supply (BioRad). Thenitrocellulose membrane was blocked using 3% fish gelatin (SigmaChemical, St. Louis, Mo.) in Tris buffered saline (TBS—20 mM Tris, 500mM NaCl, pH 7.5) for 1 hour while shaking at 37° C. The membrane wasdried at 37° C. and blocked in TBS containing 3% fish gelatin and thisprocess was repeated. The membrane was then probed with the mouse seraas described above. The primary antibody was diluted 1/50 in TBScontaining 1% fish gelatin, 0.05% Tween 20 and 0.2% sodium azide(Antibody Buffer). The membrane was incubated with the primary antibodysolution overnight on a shaker at room temperature. The membrane wasthen washed two times in TBS containing 0.05% Tween 20 (TTBS) andtransferred to antibody buffer containing a 1/10,000 dilution ofAlkaline phosphatase-conjugated mouse anti-bovine IgG clone BG-18(Sigma) and a 1/3000 dilution of avidin conjugated to alkalinephosphatase (BioRad). The membrane was incubated at 37° C. for 2 hourson a shaker, then washed in TTBS four times to remove unbound conjugate.The blot was resolved in substrate solution containing alkalinephosphate color reagent A and B in 1×AP color development Buffer(BioRad) for 30 min. at 37° C. on a shaker. The resulting Westernimmunoblots was documented using a BioRad GS-800 Densitometer (see FIGS.7 and 8).

FIG. 7 shows the serological response to vaccination using the rZincprotein as examined by Western blot (A). Lane A1 shows the molecularweight marker from 250 kDa-25 kDa; Lane A2 shows the Fuso-SRP Extract ofExample 4 probed with sera derived from mice vaccinated with the 250 μgrZinc vaccine of Group C. Note; the lack of reactivity in this lane (A2)clearly showing that this protein is not expressed under conditions ofiron restriction in contrast to lanes A3 and A4. These lanes were runwith the purified rZinc protein and probed with sera derived from micevaccinated with the 100 μg rZinc vaccine dose and the 250 doserespectively. Both lanes show a single reactive band at the ˜81 kDaregion; clearly showing a serological response to vaccination using therZinc protein. The results of this study clearly demonstrate a doseresponse to protection. For example; when the rZinc vaccine wasadministered a single time at the 100 μg dose level; no difference wasseen in reducing the incidence and/or the size of lesions compared tothe placebo controls even with a measurable serological response to thevaccine as demonstrated by Western blot. Nevertheless; when the rZincvaccine was administered two times at the 250 μg dose level there was aclear difference in the efficacy of the vaccine in reducing both theincidence and the size of lesions; clearly demonstrating a doseresponse; as the dose increased the incidence and size of lesionsdecreased. These results clearly demonstrate that the zinc receptorprotein of Fusobacterium is an excellent immunogenic target protein thatcan offer a high degree of protection against abscessation of the liver.

When the rHemin protein of Lane A5 was probed with sera derived frommice given the 250 μg rZinc vaccine of Group C no reactivity was seen;as expected.

The western blot (B) of the Fuso-SRP Extract grown under iron depleteconditions was probed with sera derived from the combo vaccine of GroupD (10 μg Fuso-SRP Extract plus 50 μg rZinc protein). Note; multiplebands reacted in Lane B1 probed with sera derived from mice vaccinatedwith the combo vaccine. In contrast; the rZinc protein of Lane B2 wasprobed with sera derived from the combo vaccine of Group D consisting of10 μg Fuso-SRP Extract plus 50 μg rZinc protein. Please note; the singlerZinc band at the ˜81 kDa region (Lane-B2) showing immunologicalreactivity and a band in Lane B1 but with a slightly lower molecularweight than the rZinc protein of Lane-B2 with an approximate molecularweight between the 76 kDa-79 kDa region. Results clearly have shown thatthe zinc protein is not expressed under iron-deplete conditions; pleaserefer to Lane-A2; (Fuso-SRP Extract probed with sera derived from the250 μg rZinc vaccine of Group C) showing no reactivity.

The Western Blot showing the serological response to the rHemin proteinis illustrated in FIG. 8. Lane 1 shows the molecular weight marker from250 kDa-25 kDa range. Lane 2 shows the Fuso-SRP Extract of Example 4probed with sera derived from mice vaccinated with the rHemin vaccine ofGroup E. Note; the lack of reactivity in this lane (2). If conditionswere absolute the Hemin protein should have reacted with the sameprotein in the Fuso-SRP Extract since this protein is expressed underiron-restricted growth conditions; please refer to FIG. 1; lane 2showing the Hemin protein expressed under iron-restricted conditions.This lack of reactivity to the sera derived from mice vaccinated withthe 100 μg rHemin vaccine of Group F could simply be due to not enoughprotein of the Fuso-SRP Extract loaded in this lane.

Lane 3 shows the rZinc protein probed with sera derived from the 100 μgrHemin vaccine of Group F. Note the lack of reactivity in this lane (3)clearly showing that the rZinc protein has no homology to rHeminprotein. In contrast; the rHemin protein run in lanes Lanes A4 and A5probed with sera derived from the 25 and 100 μg rHemin vaccinated micereacted strongly with the purified recombinant protein in lanes A4 andA5 respectively.

Example 19 Enzyme-Linked Immunosorbent Assay (ELISA)

The immunological response to the Fuso-SRP Extract and individualrecombinant proteins after vaccination was determined by measuring theIgG titers by ELISA. In brief, the two recombinant proteins were coatedin 5M urea, 100 mM NaCl, 20 mM Sodium Phosphate Buffer and the Fuso-SRPExtract was coated in the Carbonate Coating Buffer (Sigma S8875Capsules). 100 μl of each antigen was added at 250 ng/well of a 96-wellImmulon 2HB plate and incubated overnight at 4° C. with gentleagitation. The plate was washed three times with PBS wash buffer (PBScontaining 0.05% Tween 20) followed by the addition of 200 μl/well 1%PVA/PBS and incubated at 37 degrees Celsius, gentle agitation. After onehour, the plate was washed three times with PBS wash buffer. 100 μl ofPVA/PBS was placed into columns 2-11, all rows. Serial 4 fold dilutionsof the primary antisera were performed in the plate by the addition of133 μl of a 1:100 dilution to rows 1 and 12, mixing 3-4 times, withtransfer of 33 μl to the next row, towards the center of the plate for atotal of 6 dilutions for each sample. The plate was incubated for 1 hourat 37 degrees Celsius followed by three washes and addition of 100μl/well of an HRP conjugated goat anti-mouse IgG, (H+L) chain antibody(KPL #074-1806) at a 1:10,000 dilution. After 1 hour incubation, theplate was washed three times followed by the addition of 100 μl 2component ABTS Peroxidase Substrate System (KPL 50-62-01). Color wasallowed to develop for 15 minutes. The absorbance was measured at awavelength of 405-490 nm.

Example 20 ELISA Results

The serological response to vaccination was monitored by ELISA asdescribed in Example 19. Blood samples were taken at the time firstvaccination (pre-immune); second vaccination and 24 hours pre-challenge.Individual blood samples were equally pooled and analyzed by ELISA todetermine the serological response to vaccination. FIG. 9 shows theserological response to the rZinc protein and FIG. 11 illustrates theresponse to the rZinc protein in combination with the addition of theFuso-SRP Extract. First; note the amnestic response of the 250 μg rZincvaccinated group. The results show an increasing titer from firstvaccination to second vaccination in contrast to the placebo controlsand the 100 μg rZinc vaccinated group. The lack of antibody response inthe placebo controls shows that there was no pre-exposure to thisprotein. Now; in this study mice in the rZinc protein at the 100 μg doselevel inadvertently received only one vaccination; resulting in a lackof any secondary immune response as seen in FIG. 9. This lack ofsecondary immunity clearly effected the overall efficacy of this group;since there was no difference in the reduction in the incidence and sizeof lesions compared to the non-vaccinated controls. In comparison; micevaccinated with the combo vaccine consisting of 10 μg of the Fuso-SRPExtract plus 50 μg of the rZinc protein showed an immune response tovaccination with a very slight secondary response as shown in FIG. 9;yet this group showed the highest degree of efficacy in reducing theincidence and size of lesions. These results suggest that efficacy isnot completely antibody mediated and that protection from infection maybe also influenced by a non-defined cell-mediated immune response. It isinteresting to speculate that the addition of the rZinc protein to theFuso-SRP Extract may induce some type of immune-modulative effect on theimmune response.

FIG. 10 shows the serological response in mice vaccinated with therHemin at the 25 μg and 100 μg vaccine dose levels compared tonon-vaccinated controls. Please note; the antibody response tovaccination with an increase in antibody titer from first vaccinationfollowed by an amnestic response following second vaccination FIG. 10.The results showed a reduction in the incidence and size of lesions inmice vaccinated with the rHemin protein at both the 25 μg and 100 μgvaccine dose levels compared to controls. Numerically there was asignificant reduction but was not statistically significant by FisherExact. Results may have been different if a more rigorous dose findingregiment would have been done; for example by increasing the dose to 250μg as done in the rZinc protein of Group-C as defined in Table 6.

FIG. 11 shows the antibody response of the Fuso-SRP Extract at the 10and 100 μg vaccine dose levels along with the combo vaccine consistingof 50 rZinc protein plus 10 μg of the Fuso-SRP Extract. All vaccineformulations showed both a primary and secondary antibody responsefollowing vaccination. This antibody response seemed to correlate wellwith high achievement of efficacy in all vaccinated groups; see summaryTable 7. All of the Fuso-SRP Extract groups had the highest percentagein the Protected Fraction.

Example 22 Expression of Novel Hemin Proteins with the Addition of Heminto Iron Restricted Fermentation Media

The 1694 culture of example 1 was inoculated into 20 ml mTSB andincubated overnight at 37° C. in an anaerobic chamber. A 2.5 mg/mlsolution of hemin was prepared by adding 0.05 g of hemin (Sigma, StLouis, Mo.) to 20 ml of 0.1 Normal Sodium Hydroxide solution andvortexed to mix. The solubilized hemin was then sterilized through a 0.2micron filter into a sterile 50 ml conical tube. Three sets of mTSB wereprepared according to Table 9.

TABLE 9 Media Base Hemin 2,2′ bipyridyl Formulation medium concentrationconcentration FeCl3 A mTSB 20 ug/mL 15 ug/mL B mTSB 0 20 ug/mL C mTSB 00 20 ug/mL2,2′ bipyridyl was added to medium A at 15 ug/ml and autoclaved for 30minutes at 121° C. The sterile hemin solution was added to media A at800 uL per 100 mL for a final concentration of 20 ug/ml. Media Bcontained no hemin and 20 ug/ml 2,2′ bipyridyl. Medium C contained nohemin or bipyridyl, but contained FeCl₃ at 20 ug/ml.

Five mL of the overnight culture was transferred to 100 mL of eachmedium A, B and C and incubated anaerobically for 7 hours at 37° C.After 7 hours, 25 ml of the cultures were transferred to fresh 500 mlvolumes of their respective media and allowed to incubate overnight at37° C. The following morning, strong growth was observed as measured byvisual turbidity. All cultures were then centrifuged for 20 minutes at7,500×G. The supernatant was decanted and discarded. The cell pellet wasresuspended in 35 ml sterile Tris buffered water with 0.93 g/l EDTAsalt. The cell suspension was then frozen at −80° C. for a minimum of 2hours. The bacterial cell suspensions were disrupted by sonication for90 seconds at 4° C. using a Branson 450 equipped with a half inchdisruption horn (Branson, Danbury Conn.). The disrupted bacterialsuspensions were clarified by centrifugation at 39,000×g for 20 minutes.The supernatants were collected and solubilized by the addition ofsodium lauroyl sarcosinate (1% vol/vol) at 4° C. for 18 hours. Thedetergent-insoluble protein-enriched fractions were collected bycentrifugation at 39,000×g for 2.5 hours at 4° C. The protein pelletswere resuspended in 200 μl Tris-buffer (pH 7.2) and stored at −90° C.

The iron-restricted hemin-supplemented SRP extract, the iron-restrictedSRP extract and the iron-supplemented SRP Extract were subjected toelectrophoresis followed by western blot analysis with the mouse serataken 24 hours pre-challenge as described in Example 16, and with thecalf convalescent sera described in Example 7. Briefly, the SRP Extractswere size-fractionated on a Criterion TGX stain free pre-cast SDS-PAGEgel (BioRad Laboratories, Richmond Calif.) with a 4% stacking gel and7.5% resolving gel. A 10 μl sample was combined with 10 μl of SDSreducing sample buffer (62.5 mM Tris-HCL ph 6.8, 20% glycerol, 2% SDS,5% β-mercaptoethanol) and boiled for 4 minutes. Samples wereelectrophoresed at 200 volt constant current for 44 minutes at 4° C.using a Criterion cell and model 1000/500 power supply (BioRad). Bandmigration was visualized using broad range kaleidoscope standards(BioRad) to aid in the electro-blot transfer while biotinylatedPrecision Plus standards (BioRad) were used as molecular weightreferences on the blot, see FIGS. 12 and 13. The gel was documented byGel Doc EZ (BioRad). For Western blot analysis, proteins wereelectroblotted from the gel onto trans-blot nitrocellulose membranes(BioRad) overnight, at 4° C. at 50 V, in Towbin buffer (25 mM Tris, 192mM glycine, 20% methanol) using a BioRad Trans-Blot transfer cell and aPac 300 power supply (BioRad). The nitrocellulose membrane was blockedusing 3% fish gelatin (Sigma Chemical, St. Louis, Mo.) in Tris bufferedsaline (TBS—20 mM Tris, 500 mM NaCl, pH 7.5) for 1 hour while shaking at37° C. The membrane was dried at 37° C. and blocked in TBS containing 3%fish gelatin and this process was repeated. The membrane was then probedwith the mouse or calf sera as described above. The primary antibody wasdiluted 1/50 in TBS containing 1% fish gelatin, 0.05% Tween 20 and 0.2%sodium azide (Antibody Buffer). The membrane was incubated with theprimary antibody solution overnight on a shaker at room temperature. Themembrane was then washed two times in TBS containing 0.05% Tween 20(TTBS) and transferred to antibody buffer containing a 1/10,000 dilutionof Alkaline phosphatase-conjugated mouse anti-bovine IgG clone BG-18(Sigma) and a 1/3000 dilution of avidin conjugated to alkalinephosphatase (BioRad). The membrane was incubated at 37° C. for 2 hourson a shaker, then washed in TTBS four times to remove unbound conjugate.The blot was resolved in substrate solution containing alkalinephosphate color reagent A and B in 1×AP color development Buffer(BioRad) for 30 min. at 37° C. on a shaker. The resulting Westernimmunoblots were documented using a BioRad GS-800 Densitometer (seeFIGS. 12 and 13).

The SDS-PAGE showing the upregulation of the rHemin protein and thehemagglutinin protein is illustrated in FIG. 12. Lanes 1 and 5 show themolecular weight markers from 250 kDa-25 kDa range. Lane 2 shows theiron-restricted and hemin supplemented Fuso-SRP Extract from formulation(A) described in table 9. Note the upregulation of the rHemin protein atapproximately 84 kDa, and a second protein, hemagglutinin, atapproximately 150 kDa.

Lane 3 shows the iron restricted formulation B of table 9. Note the lackof expression of these two proteins in the presence of iron restrictionalone without hemin supplementation. Lane 4 shows the iron repleteformulation C of table 9. Note the lack of expression of the rHemin andhemagglutinin proteins in the presence of ferric iron. This demonstratesthat iron restriction alone is not enough to upregulate these proteins;only by limiting iron and adding back hemin as an iron source are theseproteins expressed.

In addition to the upregulation of the rHemin protein, a protein of ˜150kDa by SDS-PAGE was shown to be upregulated in the presence of hemin inan otherwise iron deplete media (FIG. 12, Lane 2). This protein wasshown to be immuno-reactive in a western blot against convalescent serafrom an experimentally challenged calf of Example 7 as illustrated inFIG. 13 at Lane 2. The closest outer membrane protein found in theannotated genome of 1694 was a hypothetical protein of 154 kDa. Thissequence was used to BLAST known sequences, and was a 100% match tofilamentous hemagglutinin. The nucleotide sequence and amino acidsequence identified is shown in FIG. 47 (SEQ ID NOs: 78 and 53,respectively).

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference in their entirety.Supplementary materials referenced in publications (such assupplementary tables, supplementary figures, supplementary materials andmethods, and/or supplementary experimental data) are likewiseincorporated by reference in their entirety. In the event that anyinconsistency exists between the disclosure of the present applicationand the disclosure(s) of any document incorporated herein by reference,the disclosure of the present application shall govern. The foregoingdetailed description and examples have been given for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed, for variations obvious to one skilled in the art will beincluded within the invention defined by the claims.

Unless otherwise indicated, all numbers expressing quantities ofcomponents, molecular weights, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless otherwise indicated to thecontrary, the numerical parameters set forth in the specification andclaims are approximations that may vary depending upon the desiredproperties sought to be obtained by the present invention. At the veryleast, and not as an attempt to limit the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. All numerical values, however, inherently contain a rangenecessarily resulting from the standard deviation found in theirrespective testing measurements.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1-30. (canceled)
 31. A composition comprising an isolated polypeptidehaving at least 85% similarity to amino acids 63-423 of SEQ ID NO:2; anda pharmaceutically acceptable adjuvant.
 32. The composition of claim 31further comprising: isolated polypeptides having molecular weights of 92kDa to 79 kDa, 73 kDa to 63 kDa, 62 kDa to 58 kDa, and 57 kDa to 47 kDa,wherein the polypeptides are isolatable from a Fusobacterium necrophorumwhen incubated in media comprising an iron chelator and not isolatablewhen grown in the media without the iron chelator, isolated polypeptideshaving molecular weights of 108 kDa to 98 kDa and 79 kDa to 69 kDa,wherein the polypeptides are isolatable from a Fusobacterium necrophorumwhen incubated in media comprising an iron chelator, are expressed bythe Fusobacterium necrophorum when incubated in media without the ironchelator and expressed at an enhanced level during growth in mediacomprising an iron chelator; and a pharmaceutically acceptable adjuvant.33. The composition of claim 31 further comprising a protein having atleast 85% similarity to amino acids 63-714 of SEQ ID NO:4.
 34. Thecomposition of claim 31 further comprising a protein having at least 85%similarity to amino acids 63-736 of SEQ ID NO:6.
 35. The composition ofclaim 31 further comprising a protein having at least 85% similarity toamino acids 63-714 of SEQ ID NO:4. and a protein having at least 85%similarity to amino acids 63-736 of SEQ ID NO:6.
 36. A methodcomprising: administering to a subject an amount of the composition ofclaim 31 effective to induce the subject to produce antibody thatspecifically binds to at least one polypeptide of the composition.
 37. Amethod for treating an infection in a subject, the method comprising:administering an effective amount of the composition of claim 31 to asubject having or at risk of having an infection caused by aFusobacterium spp.
 38. A method for treating a symptom in a subject, themethod comprising: administering an effective amount of the compositionof claim 31 to a subject having or at risk of having an infection causedby a Fusobacterium spp.
 39. A method for decreasing colonization in asubject, the method comprising: administering an effective amount of thecomposition of claim 31 to a subject colonized by or at risk of beingcolonized by a Fusobacterium spp.
 40. A method for treating an infectionin a subject, the method comprising: administering an effective amountof a composition to a subject having or at risk of having an infectioncaused by a Fusobacterium spp., wherein the composition comprisesantibody that specifically binds to a polypeptide of the composition ofclaim
 31. 41. A method for treating a symptom in a subject comprising:administering an effective amount of a composition to a subject havingor at risk of having an infection caused by a Fusobacterium spp.,wherein the composition comprises antibody that specifically binds tothe polypeptide of the composition of claim
 31. 42. A method fordecreasing colonization in a subject, the method comprising:administering an effective amount of a composition to a subjectcolonized by a Fusobacterium spp., wherein the composition comprisesantibody that specifically binds to a polypeptide of the composition ofclaim
 31. 43. The method of claim 36 wherein the subject is a mammal.44. The method of claim 43 wherein the mammal is a human, bovine, orovine.
 45. The method of claim 37 wherein the Fusobacterium spp. is F.necrophorum.
 46. The method of claim 36 wherein at least 10 micrograms(ug) and no greater than 2000 μg of polypeptide is administered.
 47. Themethod of claim 37 wherein the infection causes a condition selectedfrom metritis, hepatic abscesses, and foot rot.
 48. A kit for detectingantibody that specifically binds a polypeptide, comprising in separatecontainers: the isolated polypeptide of claim 31; and a reagent thatdetects an antibody that specifically binds the polypeptide.
 49. A kitfor detecting a polypeptide, comprising in separate containers: anantibody that specifically binds the isolated polypeptide of claim 31;and a second reagent that specifically binds the polypeptide.
 50. Acomposition comprising: isolated antibody that specifically binds to thepolypeptide of claim
 31. 51. A composition comprising: an isolated wholecell that comprises a recombinant polypeptide having at least 85%similarity to amino acids 63 through 423 of SEQ ID NO:2.